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Immunohistochemical Identification of Human Skeletal Muscle Macrophages
人骨骼肌巨噬细胞的免疫组化鉴定   

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Stefano Ciciliot Stefano Ciciliot
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Bio-protocol
Jun 2018

Abstract

Macrophages have well-characterized roles in skeletal muscle repair and regeneration. Relatively little is known regarding the role of resident macrophages in skeletal muscle homeostasis, extracellular matrix remodeling, growth, metabolism and adaptation to various stimuli including exercise and training. Despite speculation into macrophage contributions during these processes, studies characterizing macrophages in non-injured muscle are limited and methods used to identify macrophages vary. A standardized method for the identification of human resident skeletal muscle macrophages will aide in the characterization of these immune cells and allow for the comparison of results across studies. Here, we present an immunohistochemistry (IHC) protocol, validated by flow cytometry, to distinctly identify resident human skeletal muscle macrophage populations. We show that CD11b and CD206 double IHC effectively identifies macrophages in human skeletal muscle. Furthermore, the majority of macrophages in non-injured human skeletal muscle show a ‘mixed’ M1/M2 phenotype, expressing CD11b, CD14, CD68, CD86 and CD206. A relatively small population of CD11b+/CD206- macrophages are present in resting skeletal muscle. Changes in the relative abundance of this population may reflect important changes in the skeletal muscle environment. CD11b and CD206 IHC in muscle also reveals distinct morphological features of macrophages that may be related to the functional status of these cells.

Keywords: Skeletal muscle (骨骼肌), Macrophages (巨噬细胞), Immune cells (免疫细胞), Immunohistochemistry (免疫组化), CD68 (CD68), CD11b (CD11b), CD206 (CD206), Flow cytometry (流式细胞术)

Background

Macrophages are pleotropic immune cells capable of adapting to changes in the local microenvironment. Over the last several years, research has shown that macrophage phenotype is dynamic, existing on a continuum (Mosser and Edwards, 2008; Italiani and Boraschi, 2014; Martinez and Gordon, 2014). However, to date macrophage populations continue to be described using the restrictive M1 and M2 classifications. It is commonly accepted that these designations are an oversimplification of macrophage phenotype and represent opposite extremes of a continuum (Mosser and Edwards, 2008; Gordon et al., 2014; Italiani and Boraschi, 2014; Martinez and Gordon, 2014; Murray et al., 2014). M1 macrophages are classically activated, have pro-inflammatory functions and are involved in host responses to pathogens and tissue injury. M2 macrophages are alternatively activated, exhibit anti-inflammatory functions and are involved in wound healing and tissue repair. In addition to the functional definition of M1 and M2, cell surface markers have been identified to distinguish between these populations. Surface markers associated with M1 macrophages include CD40, CD64 and the co-stimulatory molecules CD80/CD86 (Lolmede et al., 2009; Ambarus et al., 2012), whereas M2 macrophages have been shown to express high levels of CD163, CD206 and galactose receptors (Lolmede et al., 2009; Ambarus et al., 2012; Roszer, 2015).

From tissue to tissue, macrophage populations are heterogeneous adopting different functional roles depending on the local environment (Gordon et al., 2014; Italiani and Boraschi, 2014). This, coupled with the macrophage continuum, has led to inconsistencies with regard to identification and nomenclature across fields and across species (Murray et al., 2014). Skeletal muscle macrophages have primarily been studied in rodent models of injury, where the M1 versus M2 macrophage classification has proven useful (Smith et al., 2008; Chazaud et al., 2009; Tidball and Villalta, 2010; Kharraz et al., 2013; Novak and Koh, 2013; Saclier et al., 2013b; Rigamonti et al., 2014; Tidball et al., 2014; Wang et al., 2014; Sciorati et al., 2016; Varga et al., 2016; Mackey and Kjaer, 2017). The skeletal muscle response to injury is characterized by highly orchestrated temporal processes. Initially, M1 macrophages phagocytize damaged skeletal muscle fibers and debris, followed by M2 macrophage-facilitated repair and regeneration (Chazaud et al., 2009; Tidball and Villalta, 2010; Kharraz et al., 2013; Saclier et al., 2013a; Tidball et al., 2014; Sciorati et al., 2016). Recent work nicely details fiber repair in human skeletal muscle in vivo, showing the presence of macrophages, using a pan-macrophage intracellular marker (CD68+), in regenerating zones along injured fibers (Mackey and Kjaer, 2017). Direct interaction between macrophages and satellite cells (Dumont and Frenette, 2013; Ceafalan et al., 2017; Du et al., 2017; Wehling-Henricks et al., 2018), and defects in skeletal muscle regeneration in the absence of macrophage participation (Arnold et al., 2007; Melton et al., 2016), highlight the necessity of these cells for skeletal muscle repair. In vitro, M1 macrophages promote skeletal muscle cell proliferation and M2 macrophages promote differentiation, suggesting that macrophages may play a role in skeletal muscle growth adaptations, as well as repair (Arnold et al., 2007; Saclier et al., 2013b).

Skeletal muscle is a highly adaptable tissue, able to respond to a wide range of external stimuli, such as exercise, inactivity, hormones and nutritional signals. In contrast to the clearly defined, strongly polarizing responses elicited by acute skeletal muscle injury, the role of tissue resident macrophages during less polarizing processes, such as responses to the aforementioned stimuli, is relatively unknown. Under non-damaging exercise conditions, animal studies report an increase in macrophage populations following aerobic and resistance exercise, linked to both metabolic and growth adaptations (DiPasquale et al., 2007; Ikeda et al., 2013). However, the mechanisms by which macrophages in skeletal muscle influence training adaptations remain to be explored. It has also been reported that resident human skeletal muscle macrophage abundance is affected by aging, obesity and diabetes (Przybyla et al., 2006; Hong et al., 2009; Varma et al., 2009; Tam et al., 2012; Fink et al., 2014; Reidy et al., 2017); however, the inconsistent use of macrophage markers across studies has made the interpretation of these findings difficult. Further, the applicability of the distinctive M1/M2 markers of polarized macrophages to tissue resident macrophages is unclear, as surface markers may not be mutually exclusive on resident macrophages under non-polarizing conditions (Italiani and Boraschi, 2014). Thus, there is a need in the field for a standardized, validated method for identifying and quantifying macrophages in human skeletal muscle. Establishing a simple and reproducible protocol for studying muscle macrophages will aide in the characterization of their role in muscle adaptations to various stimuli, independent of injury.

Although results from studies of skeletal muscle macrophages in animal models are informative, these studies often use macrophage markers that are not directly translatable for use in humans. Even when human homologs do exist, the same surface markers in mouse and rat skeletal muscles often identify different populations in human skeletal muscles, complicating the extrapolation of findings from rodent models to human studies (Murray et al., 2014). For example, CD68 is used as a pan-macrophage marker in humans and an M1 marker in mice. There is a need in the field for a standardized, validated method for identifying and quantifying macrophages in human skeletal muscle. Taking into account the limited mass of frozen muscle tissue available for analyses from human skeletal muscle biopsies (normally in the range of 100 mg), an immunohistochemical method is the most feasible approach to identifying and quantifying human skeletal muscle macrophage populations.

A variety of markers have been used to characterize human macrophages by flow cytometry. The most detailed studies have been performed utilizing peripheral blood mononuclear cells (PBMCs), artificially polarized to an M1 or M2 phenotype (Martinez et al., 2006; Ambarus et al., 2012; Iqbal, 2015). These in vitro studies characterize the expression of various marker combinations on M1 and M2 macrophages and provide a good starting point for choosing markers to identify macrophage populations in frozen human skeletal muscle tissue. CD14 has been identified as a monocyte marker, expressed mainly by macrophages but also neutrophils and dendritic cells (Table 1). In blood, CD14 co-staining with CD16 is used to stratify monocytes into three subsets: classical (CD14++/CD16-), intermediate (CD14++/CD16+) and non-classical (CD14+/CD16++) (Sprangers et al., 2016; Boyette et al., 2017). It is thought that classical monocytes give rise to tissue macrophages under homeostatic conditions; however, during an inflammatory insult all monocyte populations differentiate into macrophages (Italiani and Boraschi, 2014; Sprangers et al., 2016). In tissue, CD16 is predominantly used to identify NK cells, but is also expressed on neutrophils, granulocytes, dendritic cells and some macrophage populations (Table 1). CD11b is a commonly used marker and is expressed on subsets of lymphocytes and monocytes, these include natural killer (NK) cells, granulocytes and macrophages (Table 1). CD68 is expressed by cells in the monocyte lineage, including macrophages, and is the most commonly used macrophage marker in human skeletal muscle tissue (Table 1) (Stupka et al., 2001; Beaton et al., 2002; Peterson et al., 2003; Crameri et al., 2004; Przybyla et al., 2006; Crameri et al., 2007; Mahoney et al., 2008; Mikkelsen et al., 2009; Varma et al., 2009; Paulsen et al., 2010a; Paulsen et al., 2010b; MacNeil et al., 2011; Tam et al., 2012; Chistiakov et al., 2017; Mackey and Kjaer, 2017; Reidy et al., 2017). CD68 is a member of the lysosomal/endosomal-associated membrane glycoprotein (LAMP) family of proteins, which are mainly associated with the endosomal/lysosomal compartment. Though largely intracellular, CD68 can traffic to the cell surface. Of note, other cell types have been reported to express CD68, including hematopoietic cells, fibroblasts and endothelial cells (Table 1) (Kunisch et al., 2004; Gottfried et al., 2008; Paulsen et al., 2013; Chistiakov et al., 2017). CD206, the mannose receptor, is a well-accepted macrophage marker in skeletal muscle and is widely used to identify M2 macrophage subsets (Lolmede et al., 2009; Ambarus et al., 2012; Italiani and Boraschi, 2014; Roszer, 2015), although CD206 expression by other cell types (including satellite cells) has been reported (Table 1) (Jansen and Pavlath, 2006). M2 macrophages also express CD163 (Table 1) (Lolmede et al., 2009; Ambarus et al., 2012; Roszer, 2015). CD80 and CD86 are co-stimulatory molecules expressed by antigen presenting cells upon activation and have been used to identify M1 macrophage populations (Table 1) (Mosser and Edwards, 2008; Lolmede et al., 2009; Ambarus et al., 2012). Using three grams of discarded human hamstring muscle from patients undergoing anterior cruciate ligament (ACL) reconstruction surgery, we isolated and labeled mononuclear cells with antibodies against some of the markers described above (CD11b, CD14, CD16, CD86 and CD206) and performed multichannel flow cytometry. Due to the intracellular expression of CD68, we were not able to include CD68 in flow cytometry analyses. Mononuclear cells from skeletal muscle did not express CD16, but co-expressed the other 4 markers tested (Figures 1A-1E). Thus, human skeletal muscle macrophages have a ‘mixed’ phenotype, co-expressing both M1 (CD86) and M2 (CD206) cell surface markers (Figure 1D).

Table 1. Overview of monocyte and macrophage markers



Figure 1. Flow cytometry from discarded human hamstring muscle showing co-expression of both M1 and M2 macrophage markers. Mononuclear cells isolated from human skeletal muscle express A. Both pan-monocyte markers CD11b and CD14; B. Both the M2 macrophage marker, CD206, and the pan marker, CD11b; C. Both the M1 marker, CD86, and the pan marker CD11b; D. Both the M2 marker, CD206, and the M1 marker, CD86; E. Overlay of CD206+/CD86+ populations from panel D onto CD14/CD11b flow plot shown in panel A. This overlay shows that CD206+/CD86+ macrophages (denoted in dark blue) also express the pan-monocyte markers CD11b and CD14. Red boxes indicate cells that are double positive for the markers shown.

Using these 4 cell surface antibodies against CD11b, CD14, CD86 and CD206 that label skeletal muscle resident macrophages, we sought to develop a simple and reproducible immunohistochemical method for identification of macrophages in fresh frozen human skeletal muscle sections. CD68 is a commonly used pan-macrophage marker in human skeletal muscle. However, CD68 expression is predominantly intracellular, requiring permeabilization steps to perform immunohistochemistry (IHC). These permeabilization steps lead to inconsistent results and compromise staining with additional antibodies for cell surface markers. The cell surface localization of CD11b results in better morphological definition of macrophages and more consistent staining across samples than intracellular CD68 staining. Moreover, CD11b can readily be combined with CD206 and other cell surface markers for IHC. For these reasons, we compared CD11b and CD68 staining, and found CD11b to be comparable to CD68 as a pan-macrophage marker in human skeletal muscle (Figures 2A-2E; CD11b and CD68 antibodies cannot be used to label sections simultaneously for technical reasons, see General Note 12). Furthermore, distinct morphological features of muscle macrophages that may be related to functional status was revealed through CD11b and CD206 double IHC (Figures 8A-8C) (Durafourt et al., 2012; McWhorter et al., 2013). We describe here a detailed method for combined IHC using CD11b and CD206 antibodies on frozen human skeletal muscle sections. We also describe in detail our approach to quantifying macrophage subsets in non-injured skeletal muscle. We find the majority of human muscle macrophages are CD11b+/CD206+, whereas a small subset are CD11b+/CD206- (Figures 4A-4C). We were unable to obtain IHC results with antibodies against M1 cell surface markers (CD80 or CD86). Of note, anti-CD163 works well on frozen human skeletal muscle and can be used in place of CD11b and in combination with CD206 with this protocol (see General Note 13). Moreover, combining Pax7 (a satellite cell marker) IHC with CD206 shows very little co-expression, supporting the conclusion that CD206 co-localizes with CD11b in human skeletal muscle and is a valid macrophage marker. This protocol allows reproducible quantification of the relative abundance of CD11b+/CD206+ and CD11b+/CD206- macrophage populations in human skeletal muscle, which can be extended to human skeletal muscle adaptations, aging and disease, enabling comparison of results across studies, across labs and across diverse human populations.

Materials and Reagents

  1. Pipette tips:
    101-1,000 µl (USA Scientific, TipOne, catalog number: 1122-1832 )
    0.1-10 µl (USA Scientific, TipOne, catalog number: 1120-3812 )
  2. Gloves (VWR, catalog number: 82026-424 )
  3. Nalgene Dewar (for liquid nitrogen) (Sigma-Aldrich, catalog number: F9401 )
  4. Tri-Pour Polypropylene beaker (for cooling isopentane in liquid nitrogen) (VWR, catalog number: 89011-786)
    Manufacturer: MEDEGEN MEDICAL PRODUCTS, catalog number: PB5935-400 .
  5. MX35 Ultra Low-Profile blades for cryotomy (Thermo Fisher Scientific, catalog number: 3053835 )
  6. Superfrost Plus slides (Fisher Scientific, catalog number: 12-550-15 )
  7. Shandon Single Cytoslides (Thermo Fisher Scientific, catalog number: 5991056 )
  8. Shandon Single Cytofunnel (Thermo Fisher Scientific, catalog number: 5991040 )
  9. ImmEdge PAP pen (Vector Laboratories, catalog number: H-4000 )
  10. 1.5 ml microcentrifuge tubes (USA Scientific, catalog number: 1615-5599 )
  11. 15 ml conical tube (VWR, catalog number: 89039-666 )
  12. 24 x 50 mm, No. 1 coverglass (VWR, catalog number: 48393-081 )
  13. Kimwipes (KCWW, Kimberly-Clark, catalog number: 34120 )
  14. Cork stoppers for making muscle mounts (Fisher Scientific, catalog number: 07-782J )
  15. PTFE (Teflon) coated stainless steel spatula (Fisher Scientific, catalog number: 21-401-50A)
    Manufacturer: Saint-Gobain Performance Plastics, catalog number: D1069292 .
  16. #10 curved blade disposable scalpel (Sklar Surgical Instruments, catalog number: 06-3310 )
  17. Dumont #7 curved forceps (Fine Science Tools, catalog number: 11270-20 )
  18. Cryo Tongs (Thermo Fisher Scientific, catalog number: 4000388 )
  19. Tragacanth gum, powder (Sigma-Aldrich, catalog number: G1128-500G )
  20. Fisher O.C.T compound (Fisher Scientific, catalog number: 23-730-571 )
  21. Isopentane (2-methylbutane) (Merck, catalog number: MX0760-1 )
  22. Liquid nitrogen (Scott Gross, catalog number: SG #347 )
  23. Dry ice (Scott Gross, no catalog number available)
  24. Ice cold acetone, stored at -20 °C (Fisher Scientific, catalog number: A18-4 )
  25. Streptavidin/Biotin blocking kit (Vector Laboratories, catalog number: SP-2002 )
  26. 2.5% normal horse serum (NHS) (Vector Laboratories, catalog number: S-2012 )
  27. 50% 1x PBS (see 21 and Recipes)/50% glycerol mounting medium (glycerol - VWR, catalog number: BDH1172-1LP ) or Vectashield (Vector Laboratories, catalog number: H-1000 )
  28. Antibodies for IHC, dilutions made with 2.5% normal horse serum or PBS (Table 2)
  29. ImmPRESS-AP Anti-Mouse IgG (alkaline phosphatase) polymer detection kit (Vector Laboratories, catalog number: MP-5402 )
  30. ImmPACT Vector Red Alkaline Phosphatase (AP) Substrate (Vector Laboratories, catalog number: SK-5105 )
  31. Antibodies for multichannel flow cytometry (Table 3)

  32. Table 2. Detailed IHC Antibody Information
    Primary Antibody
    Company
    Catalog Number
    Reactivity
    Host /Isotype
    Dilution/Diluent
    CD68
    Dako
    M0814
    Human/Mouse/Rat/Monkey
    Mouse/IgG1
    (1:100)/2.5% NHS
    CD11b
    Cell Sciences
    MON1019
    Human
    Mouse/IgG1
    (1:100)/2.5% NHS
    Purified IgG1, κ
    BD
    555746
    N/A
    Mouse/IgG1
    (1:500)/2.5% NHS
    C206
    R&D Systems
    AF2534
    Human
    Goat/IgG (polyclonal)
    (1:200)/2.5% NHS
    CD163
    Hycult Biotech
    HM2157
    Human/Monkey
    Mouse/IgG1
    (1:50)/2.5% NHS
    Pax7
    Developmental Studies Hybridoma Bank
    Pax7
    Human/Mouse/Rat
    Mouse/IgG1
    (1:100)/2.5% NHS
    Biotinylated goat anti-mouse IgG1
    Jackson ImmunoResearch
    115-065-205
    Mouse IgG1
    Goat/N/A
    (1:1,000)/2.5% NHS
    ImmPRESS –AP
    Vector Laboratories
    MP-5402
    Mouse IgG
    N/A
    Neat/no dilution
    Biotinylated rabbit anti-goat IgG
    Vector Laboratories
    BA-5000
    Goat
    Rabbit/N/A
    (1:500)/2.5% NHS
    Streptavidin HRP (SA-HRP)
    Thermo Fisher Scientific
    S911
    Biotin
    N/A
    (1:500)/2.5% NHS
    Streptavidin Alexa Fluor 594 (SA-594)
    Thermo Fisher Scientific S32356
    Biotin
    N/A
    (1:200)/2.5% NHS
    Superboost TSA Alexa Fluor 488 (TSA 488)
    Thermo Fisher Scientific
    B40953
    HRP
    N/A
    (1:500)/2.5% NHS
    ImmPACT Vector Red kit
    Vector Laboratories
    SK-5105
    Alkaline Phosphatase
    N/A
    According to manufacturer’s instructions

    Table 3. Detailed antibody information for multichannel flow cytometry
    Antigen
    Fluorophore
    Laser
    Filter/Bandpass
    Ex (nm)
    Em (nm)
    Concentration
    (µg/µl)
    µl/106 cells
    (500 µl total
    volume)
    Company
    Catalog Number
    CD11b
    Alex Fluor 488
    Blue (488)
    530/30
    490
    525
    0.4*
    25
    BioLegend
    301317
    CD14
    Pacific Blue
    Violet (407)
    450/50
    410
    455
    0.5
    20
    BioLegend
    325615
    CD16
    VioGreen Violet (407)
    Violet (407)
    525/50
    405
    520
    NA
    50
    Miltenyi Biotec
    130-113-959
    CD86
    Phycoerythrin (PE)
    Blue (488)
    575/26
    496
    578
    0.1*
    25
    BioLegend
    305405
    CD206
    PerCP/Cy5.5
    Blue (488)
    695/40
    482
    695
    0.1*
    25
    BioLegend
    321121
    LIVE/DEAD
    Fixable Blue
    UV (325)
    450/50
    350
    450
    NA
    0.5
    Thermo Fisher Scientific
    L34961
    Mouse IgG1, κ
    Alex Fluor 488
    Blue (488)
    530/30
    490
    525 0.2*
    50
    BioLegend
    400132
    Mouse IgG1, κ
    Pacific Blue
    Violet (407)
    450/50
    410
    455
    0.5
    20
    BioLegend
    400131
    REA Control (S)
    VioGreen Violet (407)
    Violet (407)
    525/50
    405
    520
    NA
    50
    Miltenyi Biotec
    130-104-608
    Mouse IgG2b, κ
    Phycoerythrin (PE)
    Blue (488)
    575/26
    496
    578
    0.2
    12.5
    BioLegend
    400311
    Mouse IgG1, κ
    PerCP/Cy5.5
    Blue (488)
    695/40
    482
    695
    0.2*
    12.5
    BioLegend
    400149
    *Concentration is not specific and varies by batch

  33. LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation (Thermo Fisher Scientific, catalog number: L34961 ) (Table 3)
  34. UltraComp eBeads Compensation Beads (Thermo Fisher Scientific, catalog number: 01-2222-41 )
  35. Polyurethane ice bucket (Fisher Scientific, catalog number: 02-591-45 )
  36. 1x phosphate-buffered saline (PBS) (see Recipes)
    1. Deionized (DI) water
    2. Sodium Chloride (NaCl) (VWR, catalog number: 97061-266 )
    3. Disodium hydrogen phosphate heptahydrate (Na2HPO4·7H2O) (VWR, catalog number: 200007-704)
      Manufacturer: Acros Organic, catalog number: 206515000 .
    4. Potassium phosphate monobasic (KH2PO4) (Aldon, catalog number: PP0730-500GR )
    5. Sodium hydroxide 10 N (NaOH) (VWR, catalog number: BDH7247-1 )
    6. Hydrochloric acid, 6 N (HCl) (VWR, catalog number: 97064-758 )
  37. 30% Hydrogen peroxide, ACS, Stabilized (VWR, catalog number: BDH7690-1 ) (see Recipes)
  38. DAPI for staining cell nuclei (4',6-diamidino-2-phenylindole) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: D1306 ) (see Recipes)
  39. Rabbit polyclonal anti-Laminin (Sigma-Aldrich, catalog number: L9393 ). Use at a dilution of 1:100 in 2.5% NHS

Equipment

  1. Epifluorescent microscope with automated stage (ZEISS, model: Axio Imager M1 )
  2. Cryostat (Thermo Fisher Scientific, model: HM525 NX )
  3. P1000 pipetman (Gilson, catalog number: F123602 )
  4. P10 pipetman (Gilson, catalog number: F144802 )
  5. P2 pipetman (Gilson, catalog number: F144801 )
  6. Glass Coplin jars (VWR, catalog number: 470175-194)
    Manufacturer: VARIETY GLASS, catalog number: 674 .
  7. Humidifying slide chamber (10 slide staining tray with black lid) (Electron Microscopy Sciences, catalog number: 71396-B )
  8. Variable speed 2D rocker, 14 x 12 work surface (USA Scientific, catalog number: 2527-2000 )
  9. 4 °C refrigerator (Fisher Scientific, model: Isotemp Value Lab, catalog number: 17LREEFSA )
  10. -80 °C freezer (Thermo Fisher Scientific, model: Revco® Elite Plus )
  11. Class II Type A/B3 Biological safety cabinet (NuAire)
  12. LSR II flow cytometer equipped with a 355 nm, 405 nm and 488 nm lasers (BD Biosciences)
  13. Shandon Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific, catalog number: A78300003 )

Software

  1. Image capture software (Zeiss, Zen blue)
  2. Image processing software with an event count tool (Zeiss, Zen blue or Zen lite)
  3. Prism7 or equivalent graphing software (GraphPad)
  4. FlowJo v10 (FlowJo)

Procedure

  1. Multichannel flow cytometry validation of immunohistochemistry (IHC) macrophage markers
    1. Approximately 3 g of human skeletal muscle tissue is cleaned from a patient’s hamstring tendon that is being utilized for ACL reconstruction surgery (performed in the operating room by trained medical personnel).
    2. Place skeletal muscle tissue in an ice bucket, on ice for transport back to the laboratory, then place inside a UV sterilized biosafety cabinet and perform all possible remaining steps inside the biosafety cabinet.
    3. Split the skeletal muscle specimen into multiple chunks as needed (1-2 g each) to allow for optimal tissue digestion.
    4. Isolate skeletal muscle mononuclear cells according to the established protocol referenced here (Liu et al., 2015).
    5. After obtaining a single cell suspension, centrifuge at 500 x g (rcf) (relative centrifugal force or g-force) for 5 min at 4 °C with gentle braking to pellet cells.
    6. Wash the cell pellet by resuspending with 1 ml sterile (autoclaved) 1x PBS, at this step, cell suspensions from multiple muscle chunks can be recombined back into one sample if desired.
    7. Repeat Step A5 to pellet cells and resuspend in 500 µl of fluorophore-conjugated antibody cocktail (Table 3).
    8. Mix well by pipetting up and down 6-8 times and incubate at 4 °C in the dark for 1 h.
    9. Centrifuge at 500 x g (rcf) for 5 min at 4 °C with gentle braking to pellet cells, then resuspend in sterile 1x PBS to wash.
    10. Repeat Step A9 for a second wash then resuspend cell pellet in 1 ml sterile 1x PBS for FACS.
    11. Store in an ice bucket, on ice until fluorescence-activated cell sorting (FACS).
      1. Incubate 1 drop of UltraComp eBeads Compensation Beads with each individual antibody and follow the manufacturer’s protocol to set single channel compensation parameters for each fluorophore-conjugated antibody.
      2. An unstained sample of cell suspension should always be run to determine the location of negative cell populations (Liu et al., 2015).
      3. Determine the dead cells by LIVE/DEAD Fixable blue staining and exclude them from analyses by gating on live cells only.
      4. Set a gate excluding doublets (Liu et al., 2015) using the forward scatter height (y-axis) and forward scatter area (x-axis).
      5. Isotype control antibodies in the appropriate fluorophores are used as a control to ensure antibody staining specificity (Table 3).
    12. FACS sort CD11b+/CD14+ cells for immunocytochemistry (ICC) and collect into 1x PBS.
    13. Following the sort, pellet cells by centrifugation at 500 x g for 5 min at 4 °C and resuspend in 600 µl of 1x PBS.
    14. Load 200 µl of cell suspension into a Shandon Single Cytofunnel and centrifuge with a Shandon Cytospin 4 at 1,000 revolutions per minute (RPM) for 3 min to place cells onto Shandon Single Cytoslides.
    15. Use PAP pen to trace the circle printed on the slide indicating where the sample is located and allow slides to air dry on the bench at room temperature (RT) for 30 min.
    16. Follow Steps C8-C22 of the CD11b/CD206 double IHC protocol below, replacing the primary antibody against CD11b with anti-CD68 (Step C20c) at a 1:100 dilution (Table 2).
    17. Following overnight (ON) incubation with primary anti-CD68 antibody and washes, incubate cells with ImmPRESS-AP, anti-mouse IgG (alkaline phosphatase) polymer detection kit for 1 h at RT, rocking (Table 2).
    18. Discard ImmPRESS-AP reagent and wash cells by pipetting 1x PBS into PAP pen circle, then rock for 5 min at RT. Repeat wash 3 more times for a total of 4 washes.
    19. Visualize CD68+ staining by adding alkaline phosphatase substrate using the ImmPACT Vector Red kit according to the manufacturer’s protocol (Table 2).
      1. Staining intensity can be monitored using a standard light microscope to determine the optimal amount of staining time; 10-20 min was found to be sufficient.
      2. Staining will be both chromogenic (appearing red) and fluorescent (visible using a TRITC filter cube).
    20. Repeat Step A18 (above) to wash and coverslip using 50% 1x PBS/50% glycerol.
    21. Image or store for up to a month at 4 °C in the dark (Figure 2).


      Figure 2. Validating CD11b as a pan-macrophage, cell surface marker in human skeletal muscle. A. Representative image showing IHC for the pan, intracellular macrophage marker, CD68 (green), with DAPI stained cell nuclei (blue) in human vastus lateralis muscle. Scale bar = 50 µm. B. Correlation of CD11b+ and CD68+ macrophage numbers, identified by IHC on consecutive sections from vastus lateralis muscle, n = 44 muscle samples analyzed. P value determined by Pearson’s correlation, r = Pearson’s correlation coefficient. C. CD14+/CD11b+ mononuclear cells (shown in Figure 1, panel A) were isolated by fluorescence-activated cell sorting (FACS) and stained for CD68. Positive staining is both chromogenic (red when imaged with transmitted light, bright field, BF) and fluorescent (pseudocolored pink, FL). Left image: CD68 BF; positive staining is red. Right: CD68 FL, positive staining is pink. Cell nuclei were labeled with DAPI (blue). D-E. High magnification images showing a CD68- event (D) and CD68+ cell (E) from panel C. CD68- events were small (< 10 µm) and did not appear to be monocytes/macrophages. CD68- structures appeared to stain with DAPI, suggesting that this may be cellular debris (cell nucleus without cytoplasm). Scale bars = 10 µm.

  2. Preparing IHC mounts from a Bergstrom needle human skeletal muscle biopsy (Tarnopolsky et al., 2011; Shanely et al., 2014)
    Note: This method is optional for preparing human skeletal muscle IHC mounts.

    1. Mix tragacanth gum powder and O.C.T compound with a Teflon coated spatula at a ratio of 1 part tragacanth gum powder to 1.5 parts O.C.T to make a thick paste (mounting medium).
    2. Apply mounting medium to cork and mold into a donut shape, leaving a hole in the center where the muscle specimen will be placed (Figure 3A).
    3. Following the skeletal muscle biopsy, remove the tissue from the Bergstrom needle and quickly identify skeletal muscle pieces free of fat and connective tissue.
    4. Gently separate individual pieces, choosing the largest intact pieces for IHC (~0.5-0.75 cm in length and 50-100 mg of total tissue).
    5. Trim any fat, connective tissue or accessory skeletal muscle fragments with a scalpel to clean the sample.
    6. Lay each skeletal muscle piece for IHC side by side lengthwise, with the edges on one end aligned (this will become the top of the IHC mount) and trim the other end with a #10 curved blade scalpel to align (bottom of IHC mount) (Figure 3B).
    7. Using fine-tipped forceps and the Teflon spatula, gently roll the skeletal muscle pieces together into a solid cylindrical shape (muscle cylinder).
    8. Roll the muscle cylinder onto the tip of the spatula so that the bottom (the edge you trimmed) is aligned with the tip of the spatula.
    9. Stand the muscle cylinder/spatula up so that skeletal muscle fibers are now running perpendicular to bench and the untrimmed, aligned edge (top) is facing up.
    10. Gently place the muscle cylinder into the center of the mounting medium donut, against one side of the donut and use forceps to push the muscle cylinder off of the spatula. Remove the spatula. The muscle cylinder is now perpendicular to the surface of the cork with the bottom, trimmed end on the cork and the long axis of the skeletal muscle fibers sticking up (Figure 3C).
    11. Using the spatula, work the mounting medium in to completely enclose the skeletal muscle specimen, closing gaps and leaving no air pockets between the muscle cylinder and the mounting medium (this protects the muscle specimen from freeze damage).
    12. Sculpt the top of the mounting medium so that it is flush with the top of the skeletal muscle specimen.
    13. Rapidly freeze the IHC mount by placing into liquid nitrogen cooled isopentane for 2 min.
      1. Isopentane should be a slushy consistency, with solid just beginning to form.
      2. The cork side of the mount should be facing up and the skeletal muscle specimen facing down, completely submerged in the cooled isopentane.
    14. Once frozen, use tongs to remove the IHC mount from the cooled isopentane and place on dry ice to rest for 5 min.
    15. The final IHC mount is now ready to be stored at -80 °C until you are ready to section (Figures 3D-3E).


    Figure 3. Preparing IHC mounts from human skeletal muscle. A. Representative image illustrating the donut, made from a mixture of Tragacanth powder and O.C.T. (mounting medium), described under Procedure B Step 2. B. Representative image and illustration depicting the lengthwise orientation of muscle pieces to be mounted for sectioning. C. Representative image showing the placement of a muscle cylinder into the donut made of mounting medium. Yellow arrow serves to illustrate that the muscle is positioned so that it is against one side/wall of the donut. D. Representative images of the finished IHC muscle mount (just prior to freezing); side view (left) and top view (right).

  3. Skeletal Muscle Sectioning and CD11b/CD206 Double IHC protocol
    1. Pull IHC mount from -80 °C freezer and transport to the cryostat in a Styrofoam cooler on dry ice. Set the chamber temperature in the cryostat to -25 °C and place the IHC mount into the cryostat chamber.
    2. Adhere the IHC mount to a chuck by covering the top of the chuck with O.C.T Compound, setting the cork into the O.C.T using forceps and allowing the O.C.T to harden by freezing.
    3. Allow the IHC mount to acclimate to the temperature of the chamber for 1 h prior to sectioning. The chamber temperature may need to be adjusted for each sample in order to achieve flat sections with the greatest number of fibers in cross-section.
    4. Cross-section frozen muscle mounts with the cryostat stage angle between 8° and 10°.
      1. The stage angle may also need adjusted for some samples; however, this is less common and stage angles outside of this range are not recommended.
      2. Sample should be adjusted so the top of the mount is perpendicular to the blade when sectioning. In order to obtain cross-sectional fibers, small adjustments in the angle of the chuck/mount may need to be made. Once adjustments are made, quality sections should be cut containing at least 100 muscle fibers and no more than 20% longitudinal fibers.
    5. Using the anti-roll plate to keep sections from curling, cut frozen sections at 7 µm and pick up onto charged Superfrost Plus slides.
    6. Allow sections to dry on the benchtop at RT, then circle samples to be stained using an ImmEdge PAP pen.
    7. Allow PAP pen to dry for an additional 20-30 min.
    8. Fix sections at -20 °C for 3 min by submerging slides in a Coplin jar containing ice cold acetone. Acetone should be stored at -20 °C to maintain temperature.
    9. Quickly dab slide edges onto a paper towel to drain excess acetone and transfer to a Coplin jar filled with 1x PBS at RT.
    10. Rock the slides in 1x PBS for 5 min; repeat wash step two more times for a total of three washes.
    11. Remove slides from Coplin jar, gently flick to remove excess 1x PBS, wipe the back of the slide with a paper towel and place into a humidifying slide chamber containing approximately 1.5 cm of water in the reservoir.
    12. Block endogenous peroxidases by pipetting 3% hydrogen peroxide onto the sections and allow to incubate, rocking at RT for 8 min.
    13. Gently dump the hydrogen peroxide onto a paper towel to discard, replace with 1x PBS and rock for 5 min at RT.
    14. Repeat Step C13 two more times for a total of three washes.
    15. Dump excess 1x PBS onto a paper towel, flick slide, wipe the back with a paper towel and place back in the slide chamber.
    16. Perform Streptavidin/Biotin blocking using the kit per the manufacturer’s instructions, carrying out all incubations in the humidifying slide chamber:
      1. Incubate sections with Streptavidin blocking solution for 15 min at RT.
      2. Wash briefly: two times, 2 min with 1x PBS.
      3. Incubate sections with Biotin blocking solution for 15 min at RT.
    17. Remove biotin blocking solution and replace with 1x PBS and incubate for 5 min, at RT, rocking. Repeat this wash with 1x PBS a total of three times.
    18. Dump 1x PBS onto a paper towel, flick slide to remove excess 1x PBS, wipe the back of the slide with a paper towel and return to the slide chamber.
    19. Add enough volume of 2.5% normal horse serum (NHS) to completely cover the sections and rock at 4 °C, overnight.
    20. Remove 2.5% NHS, gently dab PAP pen with a kimwipe to re-establish hydrophobic barrier, add the primary antibody for CD11b, diluted 1:100 in 2.5% NHS, and return to the rocker at 4 °C overnight.
      1. To ensure the specificity of the primary antibody against CD11b, an isotype specific control should also be prepared by adding purified mouse IgG1, κ to a section at the same concentration as the primary antibody against CD11b (0.1 mg/ml). The isotype control listed in Table 2 has a concentration of 0.5 mg/ml and should be diluted 1:500 in 2.5% NHS and incubated overnight at 4 °C, rocking (Figure 4A).
      2. To determine signal produced by background staining of the tissue specimen, a no primary antibody control should be included. Prepare the no primary antibody control by covering the section in 2.5% NHS alone (omitting any antibody) and incubating overnight at 4 °C, rocking (Figure 4B).
      3. For identification of macrophages using CD68, substitute anti-CD68 primary antibody, diluted 1:100, for primary antibody against CD11b in this step (Table 2) (Figure 2A).
      4. For identification of specific M2 macrophage populations, substitute anti-CD163 primary antibody, diluted 1:50, for anti-CD11b primary antibody in this step and continue with the staining protocol as written (Table 2).
      5. For identification of satellite cell populations, anti-Pax7 antibody, diluted 1:100, should be substituted for primary antibody against CD11b at this step (Table 2).
    21. Remove primary antibody from sections and wash (as outlined in Step C17 above) four times, 5 min with 1x PBS, rocking at RT.
    22. Dump excess 1x PBS onto a paper towel, flick to remove remaining 1x PBS, wipe the back of the slide with a paper towel, return to the slide chamber.
    23. Add the biotinylated goat anti-mouse IgG1, diluted 1:1,000 in 2.5% NHS, and incubate by rocking for 90 min at RT.
    24. Remove the biotinylated antibody from the sections and repeat the wash in Steps C21 and C22.
    25. Add the SA-HRP, diluted 1:500 in 1x PBS, and incubate for 60 min, rocking at RT.
    26. Remove the SA-HRP from the sections and wash three times, 5 min with 1x PBS, rocking at RT.
    27. After the third wash, repeat Step C22 then add TSA 488, diluted 1:500 in 1x PBS, and incubate for 20 min, rocking at RT.
    28. Remove the TSA 488 and repeat the washes in Step C26.
    29. Repeat Steps C15-C18 to block the streptavidin and biotin used to label CD11b and prevent false co-staining.
    30. Add enough volume of 2.5% NHS to completely cover the sections and rock at RT for at least 60 min (sections can be left in 2.5% NHS longer if desired).
    31. Remove 2.5% NHS, gently dab the PAP pen with a Kimwipe to re-establish the hydrophobic barrier, add the primary antibody for CD206, diluted 1:200 in 2.5% NHS, then return the slide chamber to the rocker at 4 °C overnight.
      To determine signal produced by background staining of the tissue specimen, a no primary antibody control should be included at this step also. Prepare the no primary antibody control by covering the section in 2.5% NHS alone (omitting any antibody) and incubating overnight at 4 °C, rocking (Figure 4C).
    32. Remove CD206 antibody from sections and repeat Steps C21 and C22 to wash.
    33. Add biotinylated rabbit anti-goat IgG, diluted 1:500 in 2.5% NHS, and incubate for 90 min at RT, rocking.
    34. Remove the biotin from the sections and wash four times, 5 min with 1x PBS, rocking at RT.
    35. Add the SA-594, diluted 1:200 in 1x PBS, and incubate for 60 min, rocking at RT.
    36. Remove the SA-594 from the sections and wash three times, 5 min with 1x PBS, rocking at RT.
    37. Incubate sections with DAPI, diluted 1:10,000 in 1x PBS, for 10 min at RT, rocking.
    38. Remove DAPI from the sections and wash three times, 5 min with 1x PBS.
    39. Dump excess 1x PBS onto a paper towel and add enough volume of PBS/Glycerol or Vectashield mounting medium to cover the sections (20-50 µl/slide).
    40. Gently lower coverslip onto the slide, avoiding the formation of bubbles under the coverslip. Allow the mounting medium to spread by leaving the slides laying coverslip up for 5-10 min.
    41. Drain excess mounting medium from slides by standing vertical on a paper towel for 5 min.
    42. Image muscle sections or store slides at 4 °C protected from light until ready to image. Staining will last for several months if slides are stored properly at 4 °C, in the dark.


      Figure 4. Immunohistochemical controls validating the specificity of CD11b and CD206 staining. Representative images of staining controls. A) No primary antibody for CD206, B) Isotype control antibody for CD11b and C) No primary antibody for either CD11b or CD206. CD11b (green), CD206 (red) and cell nuclei/DAPI (blue). Scale bars = 100 µm. All images were acquired on serial sections from the same sample with a 20x objective using the same exposure settings. The same display adjustment was applied across all images. A. Images showing a lack of CD206 staining when the primary antibody for CD206 is not applied to the sections. This control shows the specificity of the CD206 antibody; staining is not a product of cross reactivity with reagents used to amplify CD11b or due to non-specific tissue staining. B. Lack of CD11b+ staining when an isotype control antibody is applied. Similar to panel A, this control shows the specificity of the CD11b antibody since the isotype-matched control does not produce non-specific staining. Additionally, this control shows that CD206 staining does not result from cross reactivity to CD11b reagents since no CD11b staining is present but CD206+ cells are clearly identified. C. Images show very low non-specific background staining of skeletal muscle tissue sections from the use of amplification reagents; thus positive macrophage staining can clearly be distinguished from tissue background.

Data analysis

Quantification of skeletal muscle fiber number and macrophage abundance 

  1. General guidelines for skeletal muscle macrophage analysis
    1. Sections to be analyzed for macrophage numbers should contain at least 50-100 skeletal muscle fibers.
    2. Non-specific staining is common around the edges of skeletal muscle sections; therefore, exclude the edges when counting macrophages.
    3. Do not include longitudinal skeletal muscle fibers in the area to be counted; this skews the number of macrophages/fiber.
    4. Areas containing blood patches, edema or fibrosis will sometimes be observed in damaged tissues (Figure 5B). These areas are usually filled with macrophages and shouldn’t be included in the same analysis as macrophages located in between skeletal muscle fibers (Figure 5A). Whether these macrophages should be quantified depends on the aim of the study.


    Figure 5. Resident macrophages largely co-express CD11b and CD206 in human vastus lateralis muscle. A. Representative images showing macrophage staining: CD11b (green), CD206 (red) and DAPI (cell nuclei, blue). The majority of macrophages are positive for both CD11b and CD206; however, CD11b+/CD206- macrophages can be observed (white arrows). Scale bars = 100 µm. B. Muscle section from the vastus lateralis showing a CD11b+/CD206+ (yellow arrow) and two CD11b+/CD206- (white arrows) monocytes. Notice in the larger field of view (at right) the CD11b+/CD206- monocytes are located in a patch of blood at the edge of the muscle section, validating CD206 as a reliable marker of tissue resident macrophages in human skeletal muscle. The patch of blood can be identified by its dried, cracked appearance and high red background signal (border between blood patch and muscle fibers in the section denoted with a dashed line). The gray X is marking an area with green background signal produced by a bubble underneath the section (further outlined in Figure S1). Scale bars = 50 µm. C. Bar graph quantification showing the number of total macrophages (all CD11b+) and CD11b+/CD206+ macrophages per 100 skeletal muscle fibers. Sixty-five muscle biopsies from the vastus lateralis were analyzed. Each dot represents macrophage counts from a single subject/section with overlap between Total CD11b+ and CD11b+/CD206+ groups, illustrating that the majority of macrophages (Total CD11b+) also express CD206 (CD11b+/CD206+). In skeletal muscle samples approximately 82% of the total macrophage population co-express CD11b and CD206.

  2. Adjust the image display in any channel so that the background staining is visible and individual skeletal muscle fibers can be distinguished (Figures 6A-6B).
  3. Manually count the number of total skeletal muscle fibers within the region of interest using the “event” tool in Zen image capture software to demarcate individual skeletal muscle fibers (Figures 6C-6D).


    Figure 6. Image adjustment and counting of skeletal muscle fiber number within a cross-section. A. Original image, prior to manipulation of the display settings; B. The same image following adjustment of the display to increase the visible background staining, allowing for the identification and counting of skeletal muscle fibers; C. Count of individual muscle fibers, denoted by yellow Xs; D. Fibers were manually outlined to help demarcate individual skeletal muscle fiber borders (white) along with the fiber count from panel C (yellow Xs). A-D) CD11b (green), CD206 (red), cell nuclei/DAPI (blue). Scale bars = 100 µm.

  4. Adjust the image display so that CD11b+ macrophages (green) and DAPI+ cell nuclei (blue) can be clearly observed and cell shape/morphology is distinct (Figures 7A-7B).
    1. Isotype-specific staining controls for antibody specificity should be used to help determine the appropriate display adjustment for identifying true positive staining (Figure 5A).
    2. No primary antibody staining controls for non-specific binding of antibodies/amplification reagents can be used to aid in determining background tissue staining and display adjustment for counting (Figures 4B-4C).


    Figure 7. Identifying and quantifying macrophages within a skeletal muscle cross-section. A. Representative image of macrophage staining in human vastus lateralis: CD11b (green), CD206 (red) and cell nuclei/DAPI (blue). B. Display adjustment of the images in panel A showing enough tissue background staining to clearly identify macrophages from non-specific tissue staining. C. Macrophage count, demarcated with colored Xs: all CD11b+ (green Xs), all CD206+ (red Xs), CD11b+/CD206+ macrophages are marked with both a green and red X. White arrows indicate areas that appear to have positive staining and are near DAPI, but were not counted due to size and/or lack of distinct morphological features determinant of a cellular structure. Other areas were not counted due to a lack of DAPI staining (yellow arrows). Scale bars = 100 µm.

  5. Using DAPI as a cell marker, manually identify and count the total number of CD11b+ macrophages located near DAPI (further explanation in the Macrophage Counting Notes section below) (Figure 7C).
    1. Skeletal muscle macrophages are morphologically heterogeneous (Figures 8A-8D). For macrophage counting, we do not discriminate between different morphological groups (i.e., all CD11b+/CD206+ macrophages are counted together regardless of morphology) (see General Note 4).
    2. If DAPI is not present near the staining, we do not count the staining as a positive event, even if the morphology seems apparent.
    3. Staining must be large enough in size to represent a cellular structure (at least the same size as an intact nucleus, > 5 µm) – small dots/patches are usually just non-specific staining of cellular debris by the secondary antibody.
    4. In patches where individual macrophages cannot be distinguished, manually count each DAPI+ nucleus that touches macrophage staining (Figures 9A-9D).


    Figure 8. Distinct skeletal muscle macrophage morphologies. A. Representative image from the vastus lateralis muscle showing a macrophage with classic morphology. Described in other tissues, the outstretched processes of resting macrophages are thought to surveil the local environment (Olah et al., 2011; Durafourt et al., 2012). B. A CD11b+/CD206+ macrophage (top) stretching out toward a second CD11b+/CD206- macrophage (bottom). C. Round or amoeboid shaped macrophage. In other tissues, this morphology is thought to be indicative of activation and the production of inflammatory cytokines (Olah et al., 2011; Durafourt et al., 2012). A-C) Scale bars = 20 µm. D. Macrophages surround a damaged skeletal muscle fiber (indicated by the presence of central nuclei). Scale bars = 50 µm. A-D) Images were acquired as Z stacks using a 40x objective and cropped to enlarge. CD11b (green), CD206 (red), cell nuclei/DAPI (blue).


    Figure 9. Counting macrophages when individual cells cluster together. A. Representative image showing an area of macrophage staining with multiple nuclei where individual macrophages cannot be distinguished. B. The image in panel A, with outlines demarcating DAPI+ nuclei touching areas of positive macrophage staining. C. Panel B images including Xs marking each nucleus counted as positive macrophage staining. D. Original image from panel A showing the Xs in panel C marking macrophage counts without the outlines demarcating positive nuclei. A-D) CD11b (green), CD206 (red) and cell nuclei/DAPI (blue). Scale bars = 50 µm.

  6. After counting total CD11b+ macrophages, go back and manually count the number of these macrophages that also express CD206.
    1. CD11b+/CD206+ macrophages are the mixed M1/M2 skeletal muscle macrophage population.
    2. CD11b+/CD206- macrophages may represent the M1 population.
  7. To determine the number of M1 macrophages, subtract the number of CD11b+/CD206+ macrophages from the total number of macrophages expressing CD11b+. This gives you the number of macrophages only expressing CD11b+ (CD11b+/CD206-).
  8. For final numbers, divide each macrophage count by the total number of fibers in the region counted.

Notes

  1. General Notes
    1. Collection of skeletal muscle biopsies was carried out in accordance with the Declaration of Helsinki. Skeletal muscle biopsies from three separate studies conducted at the University of Kentucky, the University of Alabama at Birmingham and the Geriatric Research Education and Clinical Center, Central Arkansas Veterans Healthcare System were used in this protocol. Subjects at the University of Kentucky provided their written informed consent from protocols approved by the Institutional Review Board and the University of Kentucky. Details regarding subject consent and protocol approval for the other two studies are outlined in the following publications: (Dennis et al., 2015; Long et al., 2017).
    2. We have used this protocol to identify macrophage populations in subjects ranging from age 19 to 83 and spanning a wide range of activity levels. We have found the percentage of total CD11b+ macrophages that co-express CD206 (~82%) remains relatively consistent across demographics; however, the abundance (number/fiber) and phenotype may be affected by age, obesity and/or exercise.
    3. We acknowledge that surface marker expression is not sufficient to infer function and further analyses should be employed to determine functional characteristics of macrophage populations.
    4. Skeletal muscle macrophages display heterogeneous morphology; however, we do not currently include macrophage morphology as a variable in our analyses (Figure 8). Though morphology alone is likely not enough to distinguish macrophage populations, we believe macrophage morphology may be an important and telling variable for some studies and may provide further insight into macrophage phenotype beyond surface marker expression alone.
    5. Post sectioning drying time depends on the size of the sample, but 3-4 h are usually sufficient for skeletal muscle biopsies. Following drying, slides can be stored at -20 °C if staining will be done at a later time.
    6. If samples were stored at -20 °C, slides should be allowed to warm to RT for 15-20 min prior to acetone fixation.
    7. Batch controls should be included if multiple sets of staining will be done at different time points within a single study and should include at least one control and one experimental sample.
    8. For all reagents pipetted onto the slide, use enough volume to cover the sections and fill the area you created with the PAP pen, usually between 200 and 500 µl/slide.
    9. During the incubation with 3% hydrogen peroxide, you may see bubbles form on the section indicating that the peroxide is working; a lack of bubbling will not affect the staining outcome but is indicative of poor tissue quality.
    10. Following Step C26, slides can be coverslipped with PBS/Glycerol and staining can be checked prior to moving forward with the protocol. Incubate slides in a Coplin jar with 1x PBS to remove the coverslips (they will fall off), wash 2 or 3 times with 1x PBS and continue with Step C27.
    11. Draining slides post coverslip:
      1. Slides should be drained enough that mounting medium is no longer leaking from under the coverslip and the coverslip tightly adheres to the slide.
      2. Take care not to over-drain the slides; this will lead to the formation of air pockets underneath the coverslip.
      3. For long-term storage, slides can be mounted with Vectashield mounting medium.
      4. Slides can be sealed by painting the edges of the coverslip with nail polish to prevent the formation of air pockets underneath the coverslip over time.
    12. Both CD11b and CD68 antibodies are a mouse IgG1 isotype and both require amplification using Superboost Tyramide signal amplification reagents. For these reasons, co-staining of CD11b and CD68 is not possible (false positive double staining occurred).
    13. This protocol can be adapted to identify a subset of CD206+ macrophages expressing CD163 by substituting CD163 primary antibody (see Table 1) in place of CD11b primary antibody and following the remaining protocol as written.
    14. Primary antibody for Pax7 (see Table 1) can be substituted for CD11b primary antibody to label satellite cells; the remaining protocol steps are the same (Figure 10).


      Figure 10. Skeletal muscle stem cells (satellite cells) can weakly express CD206 but are easily distinguished from macrophages. A. Representative image from human vastus lateralis showing a Pax7+ satellite cell (green, B), a CD206+ macrophage (red, C) and a Pax7+/CD206weak satellite cell (D). Cell nuclei are stained with DAPI (blue). Scale bars = 100 µm. B-D. Higher magnification of the boxes from panel A. Note the overexposure of CD206 macrophage staining (C) when the intensity is adjusted so that CD206 expression in satellite cells is visible (D). Images acquired as Z stacks with a 40x objective. Scale bars = 20 µm. E. High magnification images from human vastus lateralis muscle showing a CD206+ muscle macrophage (red) in close proximity to a Pax7+ satellite cell (green). Cell nuclei are stained with DAPI and skeletal muscle fiber borders are stained with Laminin (both in blue). Images were acquired as Z stacks with a 100x oil objective. Scale bars = 20 µm.

  2. Macrophage Counting Notes
    1. Image capture should be adjusted properly for easy identification of macrophages.
      1. Exposure times should be set so that macrophage and nuclear morphology is clear and discernable and background staining is as minimally visible as possible.
      2. Images should be acquired with a 20x objective or higher magnification.
      3. For stitched images:
        1. Section boundaries are easiest to set using DAPI staining to find the edges of the tissue section.
        2. Focus points are best set using the FITC channel (CD11b staining), to ensure that macrophages are in focus with clear morphology.
    2. Staining must be located near DAPI (within 5 µm), but does not have to be directly on top of DAPI staining. If the morphology is a macrophage and it is close but not touching DAPI, we will count the event as a macrophage (Figure 7).
    3. We have found that CD163+ macrophages make-up a subset of total CD206+ macrophages. Thus, if quantifying these populations, CD206+ macrophages should be counted as the ‘parent’ macrophage population (similar to CD11b above).
    4. Establishing a set of counting parameters prior to analyzing data sets is helpful when quantifying macrophages and also helps minimize variance between counters.
    5. Choose a handful of images as a ‘guide’ counting set and use these images to set up guidelines for how macrophages will be identified and to train new counters.
      1. It is important to stay consistent between images, groups and studies with regard to the identification of macrophages.
      2. Within one data set it is better to have the same blinded counter to analyze the entire data set due to variation between counters.
      3. We have found that although absolute numbers vary between counters, overall trends with regard to increases or decreases in the abundance of macrophage populations remain consistent if basic guidelines are outlined and followed.

Recipes

  1. 1x PBS (10 mM, pH 7.4)
    1. Mix 69.68 g NaCl, 17.36 g Na2HPO4·7H2O, 2.08 g KH2PO4
    2. Stir to dissolve in DI water
    3. Dilute 10 N NaOH 1:5 with DI water to make a 2 N solution; dilute 6 N HCl 1:3 with DI water to make a 2 N solution
    4. Adjust pH of 1x PBS with 2 N NaOH or HCl
    5. Bring to a final volume of 8 L
    6. 1x PBS can be kept at RT for up to 3 months
  2. 3% hydrogen peroxide
    Dilute 30% hydrogen peroxide 1:10 in 1x PBS
  3. DAPI for staining cell nuclei
    1. Prepare a 5 mg/ml stock solution by diluting in 1x PBS
    2. Aliquot and store at -20 °C
    3. A working dilution of 1:10,000 in 1x PBS is used for labeling nuclei

Acknowledgments

This work was supported by the National Institute of Aging grant (AG046920) and by the NIH Clinical and Translational Science Award (CTSA) (UL1TR001998) at the University of Kentucky. This work also utilized de-identified samples obtained through Merit Review Award #RX0012030 to Richard A. Dennis from the US Department of Veterans Affairs (VA), Rehabilitation R&D Service. The contents do not represent the views of the VA or US Government. The authors would like to thank Sami Michaels for help with the quantification of macrophages shown in Figure 5. We would also like to thank Doug Long for recruitment of research subjects at the University of Kentucky. Multi-channel flow cytometry was collected with help from the UK Flow Cytometry & Cell Sorting core facility (www.research.uky.edu/core/flow). The UK Flow Cytometry & Cell Sorting core facility is supported in part by the Office of the Vice President for Research, the Markey Cancer Center and an NCI Center Core Support Grant (P30 CA177558) to the University of Kentucky Markey Cancer Center.

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简介

巨噬细胞在骨骼肌修复和再生中具有很好的特征。关于驻留巨噬细胞在骨骼肌动态平衡,细胞外基质重塑,生长,代谢和适应各种刺激(包括运动和训练)中的作用知之甚少。尽管在这些过程中推测了巨噬细胞的贡献,但表征非受伤肌肉中的巨噬细胞的研究是有限的,用于鉴定巨噬细胞的方法各不相同。用于鉴定人类骨骼肌巨噬细胞的标准化方法将有助于鉴定这些免疫细胞,并可用于各研究结果的比较。在这里,我们提出免疫组织化学(IHC)协议,通过流式细胞术验证,以清楚地识别常驻人类骨骼肌巨噬细胞种群。我们显示CD11b和CD206双IHC有效识别人骨骼肌中的巨噬细胞。此外,非受伤人骨骼肌中的大多数巨噬细胞表现出“混合”M1 / M2表型,表达CD11b,CD14,CD68,CD86和CD206。在休息的骨骼肌中存在相对较少的CD11b + / CD206-巨噬细胞群。这种人口的相对丰度的变化可能反映了骨骼肌肉环境的重要变化。肌肉中的CD11b和CD206 IHC也显示出巨噬细胞的不同形态学特征,这些特征可能与这些细胞的功能状态有关。

【背景】巨噬细胞是能够适应局部微环境变化的多向性免疫细胞。在过去几年中,研究表明巨噬细胞表型是动态的,存在于连续统一体中(Mosser and Edwards,2008,Italiani and Boraschi,2014,Martinez and Gordon,2014)。然而,迄今为止巨噬细胞数量仍然使用限制性M1和M2分类进行描述。人们普遍认为这些指称是巨噬细胞表型的过度简化,代表了相反的极端情况(Mosser和Edwards,2008年,Gordon等人,2014年,Italiani和Boraschi,2014年,Martinez和Gordon ,2014年,Murray等人,2014年)。 M1巨噬细胞经典活化,具有促炎功能并参与宿主对病原体和组织损伤的应答。 M2巨噬细胞被交替激活,表现出抗炎功能并参与伤口愈合和组织修复。除了M1和M2的功能定义之外,还鉴定出细胞表面标记来区分这些群体。与M1巨噬细胞相关的表面标志物包括CD40,CD64和共刺激分子CD80 / CD86(Lolmede等人,2009,Ambarus等人,2012),而已显示M2巨噬细胞表达高水平的CD163,CD206和半乳糖受体(Lolmede等人,2009,Ambarus等人,2012,Roszer,2015)。

从组织到组织,巨噬细胞群是异质的,取决于当地环境采用不同的功能作用(Gordon等,2014,Italiani和Boraschi,2014)。这与巨噬细胞连续体结合在一起,导致了不同领域和不同物种间鉴定和命名方面的不一致(Murray等人,2014年)。主要在啮齿动物模型的损伤中研究骨骼肌巨噬细胞,其中M1和M2巨噬细胞分类已被证明是有用的(Smith等人,2008,Chazaud等人,, 2009年,Tidball和Villalta,2010年,Kharraz等人,2013年,Novak和Koh,2013年,Saclier等人,2013b,Rigamonti等人< 2014年,Tidball 等人,2014年,Wang等人2014年,Sciorati 等人,2016年,Varga 等。2016年,Mackey和Kjaer,2017年)。骨骼肌对损伤的反应以高度协调的时间过程为特征。最初,M1巨噬细胞吞噬受损的骨骼肌纤维和碎片,然后是M2巨噬细胞促进的修复和再生(Chazaud等人,2009,Tidball and Villalta,2010,Kharraz等人, 2013年,Saclier等人,2013a,Tidball等人,2014年,Sciorati等人,2016年)。最近的工作很好地详细描述了人体骨骼肌纤维在体内的修复,显示巨噬细胞的存在,使用泛巨噬细胞的细胞内标记(CD68 +),沿损伤纤维再生区域(Mackey和Kjaer,2017) 。巨噬细胞和卫星细胞之间的直接相互作用(Dumont和Frenette,2013年,Ceafalan等人,2017年,Du等人,2017年,Wehling-Henricks等人.208),以及缺乏巨噬细胞参与时骨骼肌再生的缺陷(Arnold等人,2007,Melton等人,2016年) ,突出这些细胞对于骨骼肌修复的必要性。在体外,M1巨噬细胞促进骨骼肌细胞增殖,M2巨噬细胞促进分化,表明巨噬细胞可能在骨骼肌生长适应以及修复中起作用(Arnold等, 2007年,Saclier 等人,2013b)。

骨骼肌是一种高度适应性的组织,能够对各种外部刺激作出反应,如运动,不活动,激素和营养信号。与由急性骨骼肌损伤引起的明确定义的强极化反应相反,组织驻留巨噬细胞在较少极化过程(例如对前述刺激的反应)期间的作用相对未知。在非损伤性运动条件下,动物研究报道了有氧运动和抗阻运动后巨噬细胞数量的增加,其与代谢和生长适应有关(DiPasquale等人,2007,Ikeda等人, ,2013)。然而,巨噬细胞在骨骼肌中影响训练适应性的机制仍有待探索。还有报道说,常驻人类骨骼肌巨噬细胞丰度受到衰老,肥胖和糖尿病的影响(Przybyla等人,2006,Hong等人,2009,Varma 2009年,Tam等人,2012年,Fink等人,2014年,Reidy等人,2009年, ,2017);然而,不同研究中巨噬细胞标记物的不一致使用使得这些发现的解释变得困难。此外,极化巨噬细胞的独特M1 / M2标志物对组织固定巨噬细胞的适用性尚不清楚,因为在非极化条件下,驻留巨噬细胞上的表面标志物可能并不相互排斥(Italiani and Boraschi,2014)。因此,在该领域需要用于鉴定和量化人骨骼肌中巨噬细胞的标准化的,经验证的方法。建立一个简单和可重复的研究肌肉巨噬细胞的协议将有助于表征其在肌肉适应各种刺激,独立于损伤的作用。

尽管在动物模型中研究骨骼肌巨噬细胞的结果是有益的,但这些研究通常使用不能直接翻译用于人的巨噬细胞标记物。即使存在人类同源物,小鼠和大鼠骨骼肌中相同的表面标记也常常鉴定人类骨骼肌中的不同群体,使得从啮齿动物模型到人类研究的发现的外推复杂化(Murray等人, 2014)。例如,CD68被用作人类的泛巨噬细胞标志物和小鼠的M1标志物。该领域需要用于鉴定和量化人骨骼肌中巨噬细胞的标准化,验证方法。考虑到可用于人体骨骼肌活检分析的有限质量的冷冻肌肉组织(通常在100mg范围内),免疫组化方法是鉴定和定量人骨骼肌巨噬细胞群的最可行的方法。

多种标志物已被用于通过流式细胞术来表征人类巨噬细胞。已经利用外周血单核细胞(PBMC)进行了最详细的研究,所述外周血单核细胞被人工极化为M1或M2表型(Martinez等人,2006,Ambarus等人 ,2012,Iqbal,2015)。这些体外研究表征了M1和M2巨噬细胞上各种标记组合的表达,并为选择用于鉴定冷冻人骨骼肌组织中的巨噬细胞群体的标记提供了良好的起点。 CD14已被鉴定为单核细胞标志物,主要由巨噬细胞表达,但也有嗜中性粒细胞和树突细胞(表1)。在血液中,使用与CD16共染色的CD14将单核细胞分层为三个亚组:经典(CD14 ++ / CD16-),中间(CD14 ++ / CD16 +)和非经典(CD14 + / CD16 ++)(Sprangers等, ,2016年,Boyette等人,2017年)。据认为,经典的单核细胞在稳态条件下产生组织巨噬细胞;然而,在炎症损伤期间,所有单核细胞群体分化成巨噬细胞(Italiani和Boraschi,2014,Sprangers等人,2016)。在组织中,CD16主要用于鉴定NK细胞,但也表达于嗜中性粒细胞,粒细胞,树突状细胞和一些巨噬细胞群(表1)。 CD11b是一种常用的标志物,在淋巴细胞和单核细胞亚群上表达,包括自然杀伤(NK)细胞,粒细胞和巨噬细胞(表1)。 CD68由单核细胞谱系中的细胞表达,包括巨噬细胞,并且是人骨骼肌组织中最常用的巨噬细胞标志物(表1)(Stupka等人,2001,Beaton等人2002年,Peterson等人,2003年,Crameri等人,2004年,Przybyla等人,2006年, ,2007年,Crameri等人,2007年,Mahoney等人,2008年,Mikkelsen等人,2009年,Varma等人, ,2009,Paulsen等人,2010a,Paulsen等人,2010b,MacNeil等人,2011,Tam 2012年,Chistiakov等人,2017年,Mackey和Kjaer,2017年,Reidy等人,2017年)。 CD68是溶酶体/内体相关膜糖蛋白(LAMP)家族蛋白的成员,其主要与内体/溶酶体隔室相关。尽管在很大程度上是细胞内的,但CD68可以流向细胞表面。值得注意的是,其他细胞类型已有报道表达CD68,包括造血细胞,成纤维细胞和内皮细胞(表1)(Kunisch等人,2004,Gottfried等人 >,2008,Paulsen等人,2013,Chistiakov等人,2017)。 CD206是甘露糖受体,在骨骼肌中是公认的巨噬细胞标志物,广泛用于鉴定M2巨噬细胞亚群(Lolmede等人,2009,Ambarus等人 (2012),Italiani和Boraschi,2014,Roszer,2015),但其他细胞类型(包括卫星细胞)的CD206表达已有报道(表1)(Jansen和Pavlath,2006)。 M2巨噬细胞也表达CD163(表1)(Lolmede等人,2009,Ambarus等人,2012,Roszer,2015)。 CD80和CD86是激活后由抗原呈递细胞表达的共刺激分子,并已用于鉴定M1巨噬细胞群(表1)(Mosser和Edwards,2008,Lolmede等人,2009,Ambarus ,2012)。使用来自经历前交叉韧带(ACL)重建手术的患者的3克废弃的人腿肌肉,我们分离并用针对上述一些标记物(CD11b,CD14,CD16,CD86和CD206)的抗体标记单核细胞并且执行多通道流流式细胞仪。由于CD68的细胞内表达,我们不能在流式细胞术分析中包括CD68。来自骨骼肌的单核细胞不表达CD16,但共同表达其他4个测试标记(图1A-1E)。因此,人类骨骼肌巨噬细胞具有“混合”表型,共同表达M1(CD86)和M2(CD206)细胞表面标记(图1D)。

表1.单核细胞和巨噬细胞标记物的概述



图1.来自人类腿筋肌肉的流式细胞术显示M1和M2巨噬细胞标志物的共表达从人骨骼肌分离的单核细胞表达A.泛单核细胞标志物CD11b和CD14; B. M2巨噬细胞标志物CD206和泛标志物CD11b; C. M1标记,CD86和泛标记CD11b; D. M2标记,CD206和M1标记CD86; E.将来自图D的CD206 + / CD86 +群体覆盖到图A所示的CD14 / CD11b流图上。该覆盖图显示CD206 + / CD86 +巨噬细胞(以深蓝色表示)也表达泛单核细胞标记物CD11b和CD14。红色框表示所示标记双重阳性的细胞。

我们使用这4种抗CD11b,CD14,CD86和CD206的细胞表面抗体来标记骨骼肌固有巨噬细胞,旨在开发一种简单且可重复的免疫组化方法来鉴定新鲜冷冻人骨骼肌切片中的巨噬细胞。 CD68是人骨骼肌中常用的泛巨噬细胞标志物。然而,CD68表达主要是细胞内的,需要透化步骤来进行免疫组织化学(IHC)。这些透化步骤导致不一致的结果并且损害用细胞表面标记的其他抗体染色。 CD11b的细胞表面定位导致更好的巨噬细胞的形态学定义,并且跨越样品的染色比细胞内CD68染色更一致。此外,CD11b可以容易地与CD206和其他细胞表面标志物组合用于IHC。由于这些原因,我们比较了CD11b和CD68染色,发现CD11b与人类骨骼肌中的泛巨噬细胞标记CD68相当(图2A-2E;由于技术原因,CD11b和CD68抗体不能同时用于标记切片,见通用注释12)。此外,可能与功能状态有关的肌肉巨噬细胞的不同形态特征通过CD11b和CD206双重IHC显示(图8A-8C)(Durafourt等人,2012,McWhorter等人。,2013)。我们在这里描述了在冷冻人骨骼肌切片上使用CD11b和CD206抗体的组合IHC的详细方法。我们还详细描述了我们的方法来量化在非受伤的骨骼肌巨噬细胞亚群。我们发现大多数人类肌肉巨噬细胞是CD11b + / CD206 +,而一小部分是CD11b + / CD206-(图4A-4C)。我们无法获得针对M1细胞表面标记(CD80或CD86)的抗体的IHC结果。值得注意的是,抗CD163在冷冻人骨骼肌上效果良好,可用本方案代替CD11b和CD206联合使用(见通用注释13)。此外,将Pax7(卫星细胞标记)IHC与CD206结合显示非常少的共表达,这支持了CD206与人骨骼肌中的CD11b共定位并且是有效的巨噬细胞标记物的结论。该协议允许重复定量人骨骼肌中CD11b + / CD206 +和CD11b + / CD206-巨噬细胞群的相对丰度,这可以扩展到人类骨骼肌适应性,衰老和疾病,从而跨实验室跨越研究,跨越实验室跨越多样化的人群。

关键字:骨骼肌, 巨噬细胞, 免疫细胞, 免疫组化, CD68, CD11b, CD206, 流式细胞术

材料和试剂

  1. 移液器吸头:
    101-1,000μl(USA Scientific,TipOne,目录号:1122-1832)
    0.1-10μl(USA Scientific,TipOne,目录号:1120-3812)
  2. 手套(VWR,目录号:82026-424)
  3. Nalgene杜瓦(用于液氮)(Sigma-Aldrich,目录号:F9401)
  4. 三元聚丙烯烧杯(用于冷却液氮中的异戊烷)(VWR,目录号:89011-786)
    制造商:MEDEGEN MEDICAL PRODUCTS,产品目录号:PB5935-400。

  5. 用于冷冻切片的MX35 Ultra Low-Profile刀片(Thermo Fisher Scientific,目录号:3053835)
  6. Superfrost Plus幻灯片(Fisher Scientific,产品目录号:12-550-15)
  7. Shandon Single Cytoslides(Thermo Fisher Scientific,目录号:5991056)
  8. Shandon Single Cytofunnel(Thermo Fisher Scientific,目录号:5991040)
  9. ImmEdge PAP笔(Vector Laboratories,目录号:H-4000)
  10. 1.5 ml微量离心管(USA Scientific,目录号:1615-5599)

  11. 15毫升锥形管(VWR,目录号:89039-666)
  12. 24×50毫米,1号玻璃罩(VWR,目录号:48393-081)
  13. Kimwipes(KCWW,Kimberly-Clark,目录号:34120)

  14. 软木塞制作肌肉坐骑(Fisher Scientific,目录号:07-782J)
  15. 聚四氟乙烯(聚四氟乙烯)涂层不锈钢刮刀(Fisher Scientific,目录号:21-401-50A)
    制造商:Saint-Gobain Performance Plastics,产品目录号:D1069292。
  16. #10弯曲刀片一次性手术刀(斯克拉手术器械,目录号:06-3310)
  17. Dumont#7弯钳(Fine Science Tools,目录号:11270-20)
  18. Cryo Tongs(Thermo Fisher Scientific,产品目录号:4000388)
  19. 黄蓍胶,粉末(Sigma-Aldrich,目录号:G1128-500G)
  20. Fisher O.C.T化合物(Fisher Scientific,目录号:23-730-571)
  21. 异戊烷(2-甲基丁烷)(Merck,目录号:MX0760-1)
  22. 液氮(Scott Gross,目录号:SG#347)
  23. 干冰(斯科特格罗斯,没有产品目录号)
  24. 冰冷的丙酮,储存在-20°C(Fisher Scientific,目录号:A18-4)
  25. 链霉抗生物素蛋白/生物素阻断试剂盒(Vector Laboratories,目录号:SP-2002)
  26. 2.5%正常马血清(NHS)(Vector Laboratories,目录号:S-2012)
  27. 50%1xPBS(参见21和配方)/ 50%甘油封闭介质(甘油-VWR,目录号:BDH1172-1LP)或Vectashield(Vector Laboratories,目录号:H-1000)
  28. 用于IHC的抗体,用2.5%正常马血清或PBS制备的稀释液(表2)
  29. ImmPRESS-AP抗小鼠IgG(碱性磷酸酶)聚合物检测试剂盒(Vector Laboratories,目录号:MP-5402)
  30. ImmPACT载体红色碱性磷酸酶(AP)底物(Vector Laboratories,目录号:SK-5105)
  31. 多通道流式细胞术的抗体(表3)

  32. 表2.详细的IHC抗体信息


    表3.多通道流式细胞术的详细抗体信息


  33. LIVE / DEAD固定蓝色死细胞染色试剂盒,用于紫外激发(赛默飞世尔科技,产品目录号:L34961)(表3)
  34. UltraComp eBeads补偿珠(赛默飞世尔科技,产品目录编号:01-2222-41)
  35. 聚氨酯冰桶(Fisher Scientific,目录号:02-591-45)
  36. 1x磷酸盐缓冲盐水(PBS)(见食谱)
    1. 去离子(DI)水
    2. 氯化钠(NaCl)(VWR,目录号:97061-266)
    3. 磷酸氢二钠七水合物(Na 2 HPO 4·7H 2 O)(VWR,目录号:200007-704)
      制造商:Acros Organic,产品目录号:206515000。
    4. 磷酸二氢钾(KH 2 PO 4)(Aldon,目录号:PP0730-500GR)
    5. 氢氧化钠10 N(NaOH)(VWR,目录号:BDH7247-1)
    6. 盐酸6N(HCl)(VWR,目录号:97064-758)
  37. 30%过氧化氢,ACS,稳定(VWR,目录号:BDH7690-1)(见食谱)
  38. DAPI染色细胞核(4',6-二脒基-2-苯基吲哚)(Thermo Fisher Scientific,Molecular Probes TM,目录号:D1306)(见食谱)
  39. 兔多克隆抗层粘连蛋白(Sigma-Aldrich,目录号:L9393)。在2.5%NHS中以1:100的稀释度使用。

设备

  1. 具有自动舞台的落射荧光显微镜(ZEISS,型号:Axio Imager M1)
  2. Cryostat(Thermo Fisher Scientific,型号:HM525 NX)
  3. P1000移液器(Gilson,目录号:F123602)
  4. P10 pipetman(Gilson,目录号:F144802)
  5. P2 pipetman(Gilson,目录号:F144801)
  6. 玻璃Coplin罐(VWR,目录号:470175-194)
    制造商:VARIETY GLASS,目录号:674。
  7. 加湿玻片室(10个带黑色盖子的玻片染色盘)(Electron Microscopy Sciences,目录号:71396-B)
  8. 变速2D摇摆器,14 x 12工作表面(USA Scientific,目录号:2527-2000)
  9. 4°C冰箱(Fisher Scientific,型号:Isotemp Value Lab,目录号:17LREEFSA)
  10. -80°C冰箱(赛默飞世尔科技,型号:Revco <®> Elite Plus)
  11. II类A / B3生物安全柜(NuAire)
  12. 配备355 nm,405 nm和488 nm激光器(BD Biosciences)的LSR II流式细胞仪
  13. Shandon Cytospin 4 Cytocentrifuge(Thermo Fisher Scientific,目录号:A78300003)

软件

  1. 图像捕捉软件(蔡司,禅蓝色)
  2. 带有事件计数工具的图像处理软件(Zeiss,Zen Blue或Zen Lite)
  3. Prism7或同等的图形软件(GraphPad)
  4. FlowJo v10(FlowJo)

程序

  1. 多道流式细胞术验证免疫组织化学(IHC)巨噬细胞标记物

    1. 约3克人体骨骼肌肉组织从正在用于ACL重建手术的患者ha绳肌肌腱中清除(由训练有素的医务人员在手术室中进行)。
    2. 将骨骼肌组织放入冰桶中,放在冰上运回实验室,然后放入紫外线消毒的生物安全柜内,并在生物安全柜内执行所有可能的剩余步骤。
    3. 根据需要将骨骼肌标本分成多块(每块1-2g),以便获得最佳的组织消化。
    4. 根据此处提及的已建立的方案分离骨骼肌单核细胞(Liu等人,2015)。
    5. 获得单细胞悬浮液后,在4℃以500gxg(rcf)(相对离心力或重力)离心5分钟,并轻轻制动以沉淀细胞。
    6. 通过用1ml无菌(高压灭菌)的1x PBS再悬浮来清洗细胞沉淀,如果需要,在该步骤中,可以将来自多个肌肉块的细胞悬浮液重新组合回到一个样品中。
    7. 重复步骤A5以沉淀细胞并重悬于500μl荧光团缀合的抗体混合物中(表3)。

    8. 混匀,上下移液6-8次,在4°C黑暗中孵育1小时。
    9. 在4℃以500gxg(rcf)离心5分钟,轻轻制动以沉淀细胞,然后重悬于无菌1x PBS中以洗涤。
    10. 重复步骤A9第二次洗涤,然后将细胞沉淀物重悬于1ml无菌1x PBS中用于FACS。
    11. 储存在冰桶中,冰上,直到荧光激活细胞分选(FACS)。
      1. 用每个单独的抗体孵育1滴UltraComp eBeads补偿珠,并按照制造商的方案设置每个荧光团结合抗体的单通道补偿参数。
      2. 应始终运行未染色的细胞悬液样本以确定阴性细胞群的位置(Liu等人,2015年)。
      3. 通过LIVE / DEAD固定蓝色染色确定死细胞,并仅通过对活细胞进行门控来排除它们。

      4. 使用前向散射高度(y轴)和前向散射面积(x轴)设置一个不包括双峰的门(Liu等人,2015年)。
      5. 将适当荧光团中的同种型对照抗体用作对照以确保抗体染色特异性(表3)。
    12. FACS将CD11b + / CD14 +细胞分选用于免疫细胞化学(ICC)并收集到1x PBS中。
    13. 在分选之后,通过在4℃下以500μgxg离心5分钟沉淀细胞,并在600μl的1x PBS中重悬。
    14. 将200μl细胞悬液装入Shandon Single Cytofunnel中,并用Shandon Cytospin 4以1,000转/分钟(RPM)离心3分钟,以将细胞置于Shandon Single Cytoslides上。
    15. 使用PAP笔跟踪印在幻灯片上的圆圈,指示样品位于何处,并允许载玻片在室温(RT)下在台上风干30分钟。
    16. 按照以下CD11b / CD206双重IHC方案的步骤C8-C22,用1:100稀释度的抗CD68(步骤C20c)替换抗CD11b的一抗(表2)。
    17. 在与主要抗CD68抗体孵育过夜(ON)并洗涤后,在室温下用ImmPRESS-AP,抗小鼠IgG(碱性磷酸酶)聚合物检测试剂盒孵育细胞1小时,摇动(表2)。
    18. 弃去ImmPRESS-AP试剂并通过将1×PBS移液至PAP笔圈中洗涤细胞,然后在室温下摇动5分钟。重复洗3次,共洗涤4次。
    19. 根据制造商的方案(表2),使用ImmPACT Vector Red试剂盒加入碱性磷酸酶底物以显现CD68 +染色。
      1. 使用标准光学显微镜可以监测染色强度以确定最佳染色时间量;发现10-20分钟就足够了。
      2. 染色将是发色(显示红色)和荧光(使用TRITC滤光片立方体可见)。
    20. 重复步骤A18(上述)以使用50%1x PBS / 50%甘油洗涤和盖玻片。
    21. 在4°C的黑暗环境下拍摄或存放长达一个月(图2)。


      图2.验证CD11b作为人骨骼肌中的泛巨噬细胞表面标记物A.代表性图像显示具有DAPI染色细胞核的泛,胞内巨噬细胞标记物CD68(绿色)的IHC的代表性图像(蓝色)在人的股外侧肌肉。比例尺= 50μm。 B.CD11b +和CD68 +巨噬细胞数量的相关性,通过IHC鉴定来自股外侧肌的连续切片,n = 44个分析的肌肉样品。由Pearson相关性确定的P 值,r = Pearson相关系数。 C.通过荧光激活细胞分选(FACS)分离CD14 + / CD11b +单核细胞(显示于图1,图A),并对CD68进行染色。阳性染色是显色的(用透射光,明场,BF成像时为红色)和荧光(假彩色粉红,FL)。左图:CD68 BF;阳性染色是红色的。右:CD68 FL,阳性染色是粉红色的。细胞核用DAPI(蓝色)标记。 d-E。显示来自图C的CD68-事件(D)和CD68 +细胞(E)的高放大图像.CD68-事件很小(<10μm)并且看起来不是单核细胞/巨噬细胞。 CD68-结构似乎用DAPI染色,表明这可能是细胞碎片(没有细胞质的细胞核)。比例尺= 10微米。

  2. 从Bergstrom针头人骨骼肌活组织检查中制备IHC坐骨神经(Tarnopolsky等人,2011,Shanely等人,2014)
    注意:此方法对于准备人骨骼肌IHC坐骑是可选的。

    1. 将黄蓍胶胶粉和O.C.T化合物与Teflon涂覆的刮刀以1份黄蓍胶胶粉与1.5份O.C.T的比例混合以制成厚糊(固定介质)。
    2. 将安装介质应用于软木塞并模制成圆环形状,在放置肌肉标本的中心留下一个孔(图3A)。
    3. 骨骼肌活检后,从Bergstrom针头取出组织,并快速识别无脂肪和结缔组织的骨骼肌块。
    4. 轻轻地分开单个碎片,选择最大的完整碎片用于免疫组织化学(〜0.5-0.75厘米长,50-100毫克总组织)。

    5. 用手术刀修剪任何脂肪,结缔组织或附件骨骼肌肉碎片以清洁样本。
    6. 将IHC的每个骨骼肌肉块纵向并排放置,使一端的边缘对齐(这将成为IHC底座的顶部)并用#10弯曲刀片手术刀修整另一端以对齐(IHC底座的底部) (图3B)。
    7. 使用细尖镊子和特氟龙刮刀,将骨骼肌肉块轻轻滚动成实心圆柱形(肌肉圆柱体)。
    8. 将肌肉圆柱滚动到刮刀尖端,使底部(修剪的边缘)与刮刀尖端对齐。
    9. 将肌肉圆柱/刮刀向上放置,使骨骼肌纤维现在垂直于工作台运行,未修剪的对齐边缘(顶部)朝上。
    10. 轻轻地将肌肉圆筒放入安装介质面包圈的中心,靠着面包圈的一侧,并用镊子将压力棒从压舌板上推下。取出抹刀。肌肉圆柱现在垂直于底部的软木塞表面,修剪软木塞的端部和骨骼肌纤维的长轴伸出(图3C)。
    11. 使用刮刀,将安装介质加工成完全封闭骨骼肌标本,闭合间隙并在肌肉圆柱体和安装介质之间留下空气袋(这可以保护肌肉标本免受冻害)。
    12. 雕刻安装介质的顶部,使其与骨骼肌标本的顶部齐平。
    13. 放入液氮冷却的异戊烷中快速冷冻IHC固定2分钟。
      1. 异戊烷应该是泥泞的稠度,刚刚开始形成。
      2. 底座的软木侧应朝上,骨骼肌样品朝下,完全浸没在冷却的异戊烷中。
    14. 一旦冷冻,使用钳子从冷却的异戊烷中取出IHC底座,置于干冰上静置5分钟。
    15. 最终的IHC支架现在可以储存在-80°C,直到您准备切片(图3D-3E)。


    图3.从人骨骼肌制备IHC坐标A.代表性的图像显示了由黄蓍胶粉末和O.C.T.的混合物制成的甜甜圈。 (安装介质),在程序B步骤2下描述。B.代表性的图像和插图,描绘了要安装切片的肌肉块的纵向方向。 C.代表性的图像显示肌肉圆柱体放置在由安装介质制成的圆环中。黄色箭头用来说明肌肉的位置,使其抵靠甜甜圈的一面/墙壁。 D.完成IHC肌肉坐标的代表性图像(就在冻结之前);侧视图(左)和顶视图(右)。

  3. 骨骼肌切片和CD11b / CD206双IHC方案
    1. 将IHC安装架从-80°C冷冻箱中取出,并运送到干冰上的聚苯乙烯泡沫塑料冷却器中的低温恒温器中。将低温恒温器中的室温设置为-25°C,并将IHC底座放入低温恒温室中。
    2. 通过用O.C.T化合物覆盖卡盘顶部,使用镊子将软木塞放入O.C.T,并允许O.C.T通过冷冻硬化,将IHC安装座固定到卡盘上。
    3. 在切片之前,让IHC安装座适应室的温度1小时。可能需要为每个样品调整反应室温度,以获得横截面上纤维数量最多的扁平部分。
    4. 截面冻结肌肉坐标与8°和10°之间的低温恒温器载物台角度。
      1. 舞台角度可能还需要针对某些样本进行调整;然而,这是不常见的,并且不建议在此范围之外的舞台角度。
      2. 样品应进行调整,以便在切片时,安装架的顶部与刀片垂直。为了获得横截面光纤,可能需要对卡盘/安装座的角度进行小的调整。一旦进行调整,切割的质量部分应至少包含100根肌纤维和不超过20%的纵向纤维。
    5. 使用防卷板保持切片不卷曲,切下7微米的冷冻切片并取出带电的Superfrost Plus切片。
    6. 在室温下让切片在室温下干燥,然后用ImmEdge PAP笔在圆形样本上染色。

    7. 允许PAP笔干燥20-30分钟。
    8. 通过将载玻片浸没在含有冰冷丙酮的Coplin罐中,将切片在-20℃固定3分钟。丙酮应储存在-20°C以保持温度。
    9. 迅速将边缘滑到纸巾上以排出多余的丙酮,并在室温下转移到装有1x PBS的Coplin罐中。
    10. 在1x PBS中摇动幻灯片5分钟;重复洗涤两次,共三次洗涤。
    11. 从Coplin瓶中取出载玻片,轻轻轻弹以除去多余的1x PBS,用纸巾擦拭载玻片的背面,并将其置于加湿的载玻片室中,容器中含有大约1.5cm的水。
    12. 通过在切片上吸取3%过氧化氢来阻断内源性过氧化物酶,并允许孵育,在RT下摇动8分钟。
    13. 轻轻地将过氧化氢倒入纸巾丢弃,用1x PBS代替,并在室温下摇动5分钟。
    14. 重复步骤C13两次,总共进行三次洗涤。
    15. 将多余的1x PBS倒在纸巾上,轻轻滑动,用纸巾擦拭背部并放回玻片室。
    16. 按照制造商的说明使用试剂盒进行抗生蛋白链菌素/生物素阻断,在加湿玻片室中进行所有培养:

      1. 用链霉亲和素封闭液孵育切片15分钟
      2. 简要清洗:2次,1次PBS 2分钟。
      3. 在RT下用生物素阻断溶液孵育切片15分钟。
    17. 去除生物素阻断溶液并用1x PBS代替并在室温摇动5分钟。
      使用1x PBS重复洗涤3次。
    18. 将1x PBS倒在纸巾上,轻轻滑动以除去多余的1x PBS,用纸巾擦拭玻片后部并返回玻片室。
    19. 加入足够量的2.5%正常马血清(NHS)以完全覆盖切片并在4℃下摇动过夜。
    20. 取出2.5%的NHS,用kimwipe轻轻地擦拭PAP笔以重新建立疏水屏障,添加CD11b的第一抗体,在2.5%NHS中以1:100稀释,并在4℃下过夜返回摇杆。
      1. 为确保初级抗体对CD11b的特异性,还应通过将纯化的小鼠IgG1,κ加入到与针对CD11b(0.1mg / ml)的初级抗体相同浓度的切片中来制备同种型特异性对照。表2中列出的同种型对照的浓度为0.5mg / ml,应在2.5%NHS中1:500稀释,并在4°C孵育过夜,摇动(图4A)。
      2. 为了确定由组织标本的背景染色产生的信号,应该不包括一次抗体对照。通过在2.5%NHS中单独覆盖切片(省略任何抗体)并在4°C孵育过夜,摇动(图4B)来制备无一抗抗体。
      3. 为了鉴定使用CD68的巨噬细胞,在该步骤(表2)(图2A)中用CD68替代抗CD68一抗(1:100稀释)以用于抗CD11b的一级抗体。
      4. 为了鉴定特定的M2巨噬细胞群,在本步骤中用1:50稀释的抗CD163一抗替代抗CD11b一抗,并按照书面(表2)继续染色方案。
      5. 为了鉴定卫星细胞群体,在此步骤(表2)中应该用1:100稀释的抗Pax7抗体替代针对CD11b的第一抗体。
    21. 从切片中取出第一抗体并洗涤(如上述步骤C17所述)四次,用1x PBS洗5分钟,在室温下摇动。
    22. 将过量的1x PBS倒在纸巾上,轻弹以除去剩余的1x PBS,用纸巾擦拭载玻片的背面,返回到载玻片室。
    23. 加入生物素化的山羊抗小鼠IgG1,在2.5%NHS中以1:1,000稀释,并通过在室温下摇动90分钟进行孵育。

    24. 删除生物素标记的抗体,并重复步骤C21和C22中的洗涤。
    25. 加入1:500稀释的SA-HRP,孵育60分钟,在室温下摇动。
    26. 从切片中取出SA-HRP并洗3次,用1x PBS洗5分钟,在室温下摇动。
    27. 第三次洗涤后,重复步骤C22,然后加入TSA 488,在1x PBS中以1:500稀释,孵育20分钟,在室温下摇动。

    28. 移除TSA 488并在步骤C26中重复清洗
    29. 重复步骤C15-C18以阻止用于标记CD11b的链霉亲和素和生物素,并防止假共染色。
    30. 添加足够的2.5%NHS体积以完全覆盖切片并在RT下摇动至少60分钟(如果需要,切片可以留在2.5%NHS中)。
    31. 取出2.5%NHS,用Kimwipe轻轻擦拭PAP笔以重新建立疏水屏障,加入CD206的第一抗体,在2.5%NHS中以1:200稀释,然后使载玻片室在4℃下回到摇床过夜。
      为了确定由组织标本的背景染色产生的信号,在该步骤中也不应包括一次抗体对照。通过在2.5%NHS中单独覆盖切片(省略任何抗体)并在4℃孵育过夜来摇动(图4C)来制备无一抗抗体。
    32. 从部分中移除CD206抗体并重复步骤C21和C22进行清洗。
    33. 加入生物素化的兔抗山羊IgG,在2.5%NHS中以1:500稀释,并在室温孵育90分钟,摇动。
    34. 从切片中取出生物素,洗涤4次,用1x PBS洗5分钟,在室温下摇动。
    35. 添加SA-594,在1x PBS中1:200稀释,孵育60分钟,在RT摇摆。
    36. 从切片上取下SA-594,用1x PBS清洗3次,5分钟,在室温下摇动。
    37. 用DAPI孵育切片,在1x PBS中以1:10,000稀释,在RT下10分钟,摇动。
    38. 从切片中取出DAPI,并用1x PBS冲洗3次,5分钟。
    39. 将过量的1x PBS倒在纸巾上并加入足够量的PBS /甘油或Vectashield固定介质以覆盖切片(20-50μl/载玻片)。
    40. 轻轻地将盖玻片盖在玻片上,避免在盖玻片下形成气泡。
      允许安装介质铺设盖玻片的幻灯片放置5-10分钟。

    41. 在纸巾上垂直放置5分钟,从滑道中排出多余的安装介质
    42. 图像肌肉部分或商店幻灯片在4°C避光,直到准备成像。
      如果玻片在4°C,黑暗中正确存放,染色将持续数月。


      图4.免疫组化对照验证CD11b和CD206染色的特异性。 A)没有CD206的一抗,B)CD11b的同种型对照抗体和C)CD11b或CD206的一抗不存在。 CD11b(绿色),CD206(红色)和细胞核/ DAPI(蓝色)。比例尺= 100μm。所有图像都使用相同的曝光设置,从同一样品的连续部分采集20倍物镜。所有图像都应用了相同的显示调整。 A.当未将CD206的一抗应用于切片时显示缺乏CD206染色的图像。该对照显示了CD206抗体的特异性;染色不是与用于扩增CD11b或由于非特异性组织染色的试剂交叉反应的产物。 B.当应用同种型对照抗体时缺乏CD11b +染色。与A组类似,该对照显示了CD11b抗体的特异性,因为同种型匹配的对照不产生非特异性染色。另外,该对照显示CD206染色不是由与CD11b试剂的交叉反应性导致的,因为不存在CD11b染色,但是CD206 +细胞被清楚地鉴定。 C.图像显示来自使用扩增试剂的骨骼肌组织切片的非特异性背景染色非常低;因此阳性巨噬细胞染色可以清楚地区分组织背景。

数据分析

骨骼肌纤维数量和巨噬细胞丰度的定量&nbsp;


  1. 骨骼肌巨噬细胞分析的一般准则
    1. 要分析巨噬细胞数量的部分应该包含至少50-100骨骼肌纤维。
    2. 非特异性染色在骨骼肌部分的边缘周围是常见的;因此,在计数巨噬细胞时排除边缘。
    3. 不要在要计数的区域包括纵向骨骼肌纤维;这会扭曲巨噬细胞/纤维的数量。
    4. 有时会在受损组织中观察到含有血斑,水肿或纤维化的区域(图5B)。这些区域通常充满巨噬细胞,不应该与位于骨骼肌纤维之间的巨噬细胞进行相同的分析(图5A)。这些巨噬细胞是否应该量化取决于研究的目的。


    图5.显示巨噬细胞染色的代表性图像:CD11b(绿色),CD206(红色)和DAPI(细胞核,蓝色)。图5.巨噬细胞在人外侧肌中大量共表达CD11b和CD206。 。大多数巨噬细胞对于CD11b和CD206都是阳性的;然而,可以观察到CD11b + / CD206-巨噬细胞(白色箭头)。比例尺= 100μm。 B.来自股外侧肌的肌肉切片显示CD11b + / CD206 +(黄色箭头)和两个CD11b + / CD206-(白色箭头)单核细胞。注意在更大的视野中(右图),CD11b + / CD206-单核细胞位于肌肉切片边缘的一块血液中,证实CD206是人骨骼肌中组织驻留巨噬细胞的可靠标记。可以通过其干燥的裂纹外观和高红色背景信号(在用虚线表示的部分中的血斑和肌纤维之间的边界)来识别血斑。灰色的X标记了一个区域,该区域具有由该区域下方的气泡产生的绿色背景信号(进一步在图S1 )。比例尺= 50微米。 C.条形图定量显示每100个骨骼肌纤维中总巨噬细胞(所有CD11b +)和CD11b + / CD206 +巨噬细胞的数量。分析来自股外侧肌的65个肌肉活组织检查。每个点代表来自单个受试者/部分的总CD11b +和CD11b + / CD206 +组之间重叠的巨噬细胞计数,说明大多数巨噬细胞(总CD11b +)也表达CD206(CD11b + / CD206 +)。在骨骼肌样品中,大约82%的总巨噬细胞群体共表达CD11b和CD206。

  2. 调整任何通道中的图像显示,以便背景染色可见,并且可以区分单独的骨骼肌纤维(图6A-6B)。

  3. 使用Zen图像捕捉软件中的“事件”工具手动计算感兴趣区域内总骨骼肌纤维的数量,以划分单个骨骼肌纤维(图6C-6D)。


    图6.横截面内骨骼肌纤维数量的图像调整和计数A.在操作显示设置之前的原始图像; B.调整显示器后的相同图像以增加可见的背景染色,允许对骨骼肌纤维进行鉴定和计数; C.单个肌纤维的数量,由黄色Xs表示; D.手动勾画纤维以帮助划分单个骨骼肌纤维边界(白色)以及来自面板C(黄色X)的纤维计数。 A-D)CD11b(绿色),CD206(红色),细胞核/ DAPI(蓝色)。比例尺= 100μm。

  4. 调整图像显示,以便清晰地观察CD11b +巨噬细胞(绿色)和DAPI +细胞核(蓝色),并且细胞形状/形态明显不同(图7A-7B)。
    1. 应使用抗体特异性的同种型特异性染色对照来帮助确定适当的显示调整以鉴定真阳性染色(图5A)。
    2. 对于抗体/扩增试剂的非特异性结合,可以使用一级抗体染色对照来帮助确定背景组织染色和计数显示调整(图4B-4C)。


    图7.识别和量化骨骼肌横切面内的巨噬细胞A.代表人体股外侧肌巨噬细胞染色的图像:CD11b(绿色),CD206(红色)和细胞核/ DAPI(蓝色) )。 B.显示A组中图像的调整显示足够的组织背景染色以清楚地识别来自非特异性组织染色的巨噬细胞。 C.巨噬细胞计数,用有色X标定:所有CD11b +(绿色Xs),所有CD206 +(红色Xs),CD11b + / CD206 +巨噬细胞都标有绿色和红色X.白色箭头表示看起来具有阳性染色的区域,在DAPI附近,但由于尺寸和/或缺乏细胞结构的独特形态特征决定因素而未被计数。其他区域由于缺乏DAPI染色(黄色箭头)而未被计数。比例尺= 100μm。

  5. 使用DAPI作为细胞标记,手动识别并计数位于DAPI附近的CD11b +巨噬细胞总数(下文巨噬细胞计数注释 部分进一步解释)(图7C)。
    1. 骨骼肌巨噬细胞在形态学上是不均匀的(图8A-8D)。对于巨噬细胞计数,我们不区分不同的形态组(即,所有CD11b + / CD206 +巨噬细胞都统计在一起,无论形态如何)(见一般注释4)。
    2. 如果DAPI不存在于染色附近,即使形态看起来很明显,我们也不会将染色计数为阳性事件。
    3. 染色的尺寸必须足够大以代表细胞结构(至少与完整细胞核的尺寸相同,>5μm) - 小点/贴片通常只是第二抗体对细胞碎片的非特异性染色。 />
    4. 在不能区分单个巨噬细胞的斑块中,手动计数每个接触巨噬细胞染色的DAPI +细胞核(图9A-9D)。


    图8.不同的骨骼肌巨噬细胞形态A.来自股外侧肌的代表性图像显示具有典型形态学的巨噬细胞。在其他组织中描述,静止巨噬细胞的突出过程被认为是监视当地环境(Olah等人,2011,Durafourt等人,2012)。 B.向第二CD11b + / CD206-巨噬细胞伸展的CD11b + / CD206 +巨噬细胞(顶部)(底部)。 C.圆形或变形虫形巨噬细胞。在其他组织中,这种形态被认为是活化和炎性细胞因子产生的指示(Olah等人,2011,Durafourt等人,2012)。 A-C)比例尺=20μm。 D.巨噬细胞包围受损的骨骼肌纤维(由中央核的存在表示)。比例尺= 50微米。 A-D)使用40x物镜以Z堆叠的形式获取图像并裁剪放大。 CD11b(绿色),CD206(红色),细胞核/ DAPI(蓝色)。


    图9.当单个细胞聚集在一起时计数巨噬细胞A.代表性图像显示巨噬细胞染色的区域具有多个细胞核,其中不能区分单个巨噬细胞。 B.图A中的图像,其中概述了划分DAPI +细胞核接触区的阳性巨噬细胞染色。 C.包括标记每个细胞核的Xs的图B图像计数为阳性巨噬细胞染色。 D.来自图A的原始图像显示C组标记的巨噬细胞计数中的X,而没有描绘正核的轮廓。 A-D)CD11b(绿色),CD206(红色)和细胞核/ DAPI(蓝色)。比例尺= 50微米。

  6. 在计数总CD11b +巨噬细胞后,返回并手动计数也表达CD206的这些巨噬细胞的数量。
    1. CD11b + / CD206 +巨噬细胞是混合的M1 / M2骨骼肌巨噬细胞群。
    2. CD11b + / CD206-巨噬细胞可能代表M1群体。
  7. 为了确定M1巨噬细胞的数量,从表达CD11b +的巨噬细胞总数中减去CD11b + / CD206 +巨噬细胞的数量。这给你只有表达CD11b +(CD11b + / CD206-)的巨噬细胞的数量。
  8. 对于最终数字,将每个巨噬细胞计数除以该区域的纤维总数。

笔记

  1. 一般注意事项
    1. 按照赫尔辛基宣言进行骨骼肌活组织检查。来自肯塔基大学,伯明翰阿拉巴马大学和阿肯色州中部退伍军人医疗保健系统的老年研究教育和临床中心进行的三项独立研究的骨骼肌活检被用于本协议。肯塔基大学的科目由机构审查委员会和肯塔基大学批准的协议提供了书面知情同意书。关于另外两项研究的受试者同意和方案批准的细节在以下出版物中概述:(Dennis等人,2015年,Long等人,2017年)。 br />
    2. 我们已经使用这个协议来确定从19岁到83岁不等的受试者中的巨噬细胞群,并且跨越多种活动水平。我们发现共表达CD206(〜82%)的总CD11b +巨噬细胞在人口统计学上保持相对一致的百分比;然而,丰度(数量/纤维)和表型可能会受到年龄,肥胖和/或运动的影响。
    3. 我们承认表面标志物表达不足以推断功能,应进一步分析以确定巨噬细胞群体的功能特征。
    4. 骨骼肌巨噬细胞表现出非均质形态;然而,我们目前并未将巨噬细胞形态学作为变量分析(图8)。尽管单独形态学可能不足以区分巨噬细胞群,但我们相信巨噬细胞形态学可能是一些重要的研究变量,并可能进一步提供超出表面标志物表达的巨噬细胞表型的观察。
    5. 切片后干燥时间取决于样品的大小,但3-4小时通常足够用于骨骼肌活组织检查。干燥后,如果稍后进行染色,可将载玻片保存在-20°C。
    6. 如果样品储存在-20°C,在丙酮固定之前,应使载玻片升温至室温15-20分钟。
    7. 如果在一项研究中的不同时间点进行多组染色,应该包括批次控制,并应包括至少一个对照和一个实验样本。
    8. 对于移液器上移动的所有试剂,使用足够的体积来覆盖这些部分,并用PAP笔填充您创建的区域,通常在200和500μl/ slide之间。
    9. 在与3%过氧化氢孵育过程中,可能会在截面上看到气泡形成,表明过氧化氢正在工作;缺少起泡不会影响染色结果,但表示组织质量差。
    10. 在步骤C26之后,载玻片可以用PBS /甘油盖上盖片,并且在向前移动方案之前可以检查染色。在带有1x PBS的Coplin瓶中孵育载玻片以去除盖玻片(它们将脱落),用1x PBS洗涤2或3次并继续步骤C27。
    11. 排水滑盖后盖玻片:
      1. 载玻片应该足够的排空,使得载玻片不再从盖玻片下方泄漏,并且盖玻片紧紧地粘合到玻片上。
      2. 注意不要过度排出幻灯片;这将导致在盖玻片下形成气穴。
      3. 对于长期存放,幻灯片可以安装Vectashield安装介质。
      4. 可以通过用指甲油涂抹盖玻片的边缘来密封玻片,以防止随着时间的推移在盖玻片下面形成气穴。
    12. CD11b和CD68抗体都是小鼠IgG1同种型,都需要使用Superboost酪胺信号扩增试剂进行扩增。由于这些原因,CD11b和CD68的共染色是不可能的(假阳性双染色发生)。
    13. 通过用CD163第一抗体(参见表1)代替CD11b第一抗体并按照书面的剩余方案,可以修改该方案以鉴定表达CD163的CD206 +巨噬细胞的亚组。
    14. Pax7的一级抗体(见表1)可以替代CD11b一级抗体来标记卫星细胞;其余协议步骤是相同的(图10)。


      图10.骨骼肌干细胞(卫星细胞)可以弱表达CD206,但很容易与巨噬细胞区分开来。A.来自人体股外侧肌的代表性图像显示Pax7 +卫星细胞(绿色,B),CD206 +巨噬细胞(红色,C)和Pax7 + / CD206弱卫星细胞(D)。细胞核用DAPI(蓝色)染色。比例尺= 100μm。 B-d。放大A组方框的放大倍率。注意当调节强度以使卫星细胞中的CD206表达可见时,CD206巨噬细胞染色(C)的过度曝光(D)。作为Z堆叠的图像以40倍物镜获取。比例尺= 20μm。 E.来自人体股外侧肌的高倍放大图像显示紧邻Pax7 +卫星细胞(绿色)的CD206 +肌肉巨噬细胞(红色)。细胞核用DAPI染色,骨骼肌纤维边界用层粘连蛋白染色(均为蓝色)。图像是作为Z堆栈与100倍油目标获得的。比例尺= 20微米。

  2. 巨噬细胞计数笔记
    1. 应正确调整图像捕捉以便于识别巨噬细胞。
      1. 应设置暴露时间,使巨噬细胞和核形态清晰可辨,背景染色尽可能最小化。

      2. 应以20倍物镜或更高放大倍数获取图像。
      3. 对于拼接图像:
        1. 使用DAPI染色找到组织切片的边缘是最容易的部分边界。
        2. 使用FITC通道(CD11b染色)对焦点最好设置,以确保巨噬细胞聚焦清晰的形态。
    2. 染色必须位于DAPI附近(5μm以内),但不一定要直接在DAPI染色之上。如果形态学是一种巨噬细胞,并且它接近但不接触DAPI,我们将把事件计数为巨噬细胞(图7)。
    3. 我们发现CD163 +巨噬细胞构成了总CD206 +巨噬细胞的一个子集。因此,如果量化这些人群,应将CD206 +巨噬细胞计数为“亲代”巨噬细胞群(类似于上述CD11b)。
    4. 在分析数据集之前建立一套计数参数对量化巨噬细胞有帮助,并有助于减少计数器之间的差异。
    5. 选择一些图像作为“指导”计数设置,并使用这些图像为巨噬细胞如何识别以及培训新计数器设置指导。

      1. 在确定巨噬细胞方面,图像,小组和研究之间保持一致非常重要
      2. 在一个数据集中,由于计数器之间的差异,最好使用相同的盲法计数器来分析整个数据集。
      3. 我们发现,虽然计数器之间的绝对数量有所不同,但如果概述并遵循基本指导原则,则巨噬细胞数量增加或减少的整体趋势仍然保持一致。

食谱

  1. 1×PBS(10mM,pH7.4)
    1. 混合69.68g NaCl,17.36g Na 2 HPO 4·7H 2 O,2.08g KH 2 PO 4, sub> 4
    2. 搅拌溶解在去离子水中
    3. 用去离子水稀释10N氢氧化钠1:5制成2N溶液;
      用去离子水稀释6 N HCl 1:3,制成2 N溶液
    4. 用2N NaOH或HCl调节1x PBS的pH值
    5. 带来8升的最终音量
    6. 1x PBS可保存在室温下3个月
  2. 3%过氧化氢

    在1x PBS中稀释30%过氧化氢1:10
  3. DAPI染色细胞核

    1. 用1x PBS稀释制备5 mg / ml的储备溶液
    2. 分装并储存在-20°C
    3. 1×PBS中1:10,000的工作稀释度用于标记核

致谢

这项工作得到了国家老化研究所(AG046920)和美国国立卫生研究院临床和转化科学奖(CTSA)(UL1TR001998)在肯塔基大学的支持。这项工作还利用从美国退伍军人事务部(VA)Rehabilitation R&amp; D服务部的Richard A. Dennis获得的通过优异评审奖#RX0012030获得的去识别样品。内容不代表VA或美国政府的意见。作者要感谢Sami Michaels在量化图5所示的巨噬细胞方面提供的帮助。我们还要感谢Doug Long在肯塔基大学招聘研究课题。在英国流式细胞术&amp;公司的帮助下收集多通道流式细胞术。细胞分选核心设施( www.research.uky.edu/core/flow )。英国流式细胞仪&amp;细胞分选核心设施部分由研究副总裁办公室,马基癌症中心和NCI中心核心支持资助(P30 CA177558)为肯塔基大学Markey癌症中心提供支持。

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引用:Kosmac, K., Peck, B. D., Walton, R. G., Mula, J., Kern, P. A., Bamman, M. M., Dennis, R. A., Jacobs, C. A., Lattermann, C., Johnson, D. L. and Peterson, C. A. (2018). Immunohistochemical Identification of Human Skeletal Muscle Macrophages. Bio-protocol 8(12): e2883. DOI: 10.21769/BioProtoc.2883.
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