Immunohistochemistry

Macrophage identification and quantification were performed according to a detailed and validated method.59 Briefly, macrophages were identified by stepwise staining with antibodies against the pan macrophage marker CD11b (Cell Sciences) or the M2c macrophage marker CD163 (Hycult Biotech), followed by CD206, a marker of M2 macrophages (R&D Systems) (Table S1). For staining, slides were fixed in −20°C acetone and blocking steps were taken to prevent nonspecific background staining and cross‐reactivity between amplification reagents. CD11b, CD163, and CD206 labeling was amplified using a biotin, streptavidin system. For CD11b or CD163, an extra amplification step with fluorophore‐conjugated tyramide signal amplification reagent was performed. Sections were washed and nuclei were visualized using 4′,6‐diamidino‐2‐phenylindole (DAPI) (ThermoFisher). After staining, slides were coverslipped with Vectashield (Vector Laboratories, H‐1000) and stitched images of whole muscle cross‐sections were acquired with a 20× objective. The abundance of all CD11b‐expressing cells (total macrophages), CD11b+CD206−, and CD11b+CD206+ were manually quantified using the event count tool in Zen software (Zeiss). For samples with remaining tissue (n=6 non‐PAD, n=18 PAD), CD163+CD206+ macrophages were also quantified. Of note, CD163 and CD11b staining cannot be performed on the same samples because of amplification cross‐reactivity; however, the proportion of CD206+ macrophages co‐expressing CD163 was between 89% and 99.7%, supporting the idea that CD206+ macrophages are M2‐like.

A primary antibody against CD16B/FCGR3B (LifeSpan BioSciences, Inc), a neutrophil‐specific marker in human tissue, was used to assess the presence of neutrophils within muscle samples. For staining, sections were fixed for 30 minutes with Bouin fixative (Electron Microscopy Sciences, 15990), dehydrated with xylenes, and rehydrated with decreasing concentrations of ethanol. Heat‐induced epitope retrieval was performed by incubating slides in sodium citrate buffer within a heated water bath, beginning at 65°C and heating to 92°C, then incubating at 92°C for an additional 20 minutes. Slides were cooled to room temperature and washed with distilled water. Nuclei were counterstained with hematoxylin, washed in tap water, differentiated with acidified ethanol, blued in Scott water, and rinsed before blocking. Endogenous alkaline phosphatases were quenched by incubating sections for 10 minutes with Bloxall reagent (Vector Laboratories, SP‐6000). Sections were then washed, blocked for 1 hour with 2.5% normal horse serum (Vector Laboratories, S‐2012) and incubated with primary antibody for 2 hours at room temperature. CD16 antibody was amplified using ImmPRESS AP (alkaline phosphatase) anti‐rabbit polymer (Vector Laboratories, MP‐5401) and visualized by incubation with the alkaline phosphatase substrate, Vector Red (Vector Laboratories, MP‐7401), according to the manufacturer's instructions. After staining, slides were coverslipped with VectaMount AQ Aqueous Mounting Medium (Vector Laboratories, H‐5501) and representative images were acquired using transmitted light microscopy.

ECM was quantified using α‐wheat germ agglutinin (WGA), which binds to glycosaminoglycans.77 After fixation with 4% paraformaldehyde, sections were washed with PBS then incubated with Texas Red–conjugated WGA for 2 hours (ThermoFisher). Sections were washed and coverslipped with Vectashield. Stitched images of whole muscle cross‐sections were acquired with a 10× objective. Quantification of WGA was performed by setting a threshold for positive staining and determining the percent of total area in the region of interest occupied by this threshold. At least 3 regions of interest/section were analyzed using the interactive measurement wizard in AxioVision software version 4.2.8 (Zeiss). Total collagen content within the ECM was quantified with Sirius Red (SR) staining. Collagens within muscle sections were denatured by fixing for 15 minutes with Bouin fixative at 56°C in a sealed chamber within a heated water bath. Following fixation, the sealed chamber was transferred to a chemical fume hood, Bouin fixative was removed, and slides were washed with distilled water. Following the final wash, slides were incubated for 2 hours in 0.1% SR staining solution (Electron Microscopy Sciences, 26357‐02). Following staining, slides were washed in 0.5% acetic acid in distilled water, dehydrated in 95% ethanol followed by 100% ethanol, and equilibrated in xylenes. Slides were mounted with Cytoseal 60 (ThermoFisher, 8311), which was allowed to harden in a chemical fume hood for 24 hours. Whole muscle cross‐sections were imaged with a 10× objective and quantification of SR was performed similar to WGA quantification.

To visualize collagen surrounding capillaries, frozen muscle sections were dried and rehydrated for 5 minutes with 1x PBS, followed by two 5‐minute washes with PBS. Sections were blocked for 1 hour in 2.5% normal horse serum then incubated for 90 minutes with a mixture of 2 lectins: biotinylated Ulex europaeus (Vector Laboratories) and biotinylated Griffonia Simplicifolia (Vector Laboratories), and a pan‐collagen antibody (ThermoFisher). Sections were then washed and incubated for 1 hour with Streptavidin Alexa Fluor 594 (ThermoFisher) to visualize capillaries and anti‐Rabbit Alexa Fluor 488 (ThermoFisher) to visualize collagen. After 3 final washes, the slides were coverslipped with Vectashield and imaged.

Analysis of satellite cell abundance was performed as follows. After drying at room temperature, sections were fixed in −20°C acetone, washed with PBS, incubated with 3% hydrogen peroxide, and blocked for 1 hour in 2.5% normal horse serum. Satellite cells were labelled by overnight incubation with anti–paired box 7 (Pax7) (Developmental Studies Hybridoma Bank, University of Iowa). Following PBS washes, sections were incubated with biotinylated anti‐mouse IgG1 (Jackson ImmunoResearch Laboratories) for 90 minutes. Washes were repeated followed by a 1‐hour incubation with streptavidin horseradish peroxidase (ThermoFisher). Fluorescent labelling was achieved by incubating with SuperBoost tyramide signal amplification Alexa Fluor 488 in PBS (ThermoFisher) for 20 minutes. Primary antibody against laminin (Millipore Sigma) was added followed by anti‐Rabbit Alexa Fluor 594 secondary antibody (ThermoFisher) to visualize and quantify muscle fibers. Sections were washed, incubated for 10 minutes with DAPI to label nuclei, and coverslipped. Whole cross‐section images were acquired with a 20× objective, satellite cells were identified as Pax7+/DAPI+, and quantification was expressed relative to laminin‐delineated fiber number. Laminin‐stained images were previously used to determine fiber size by minimum feret diameter.69

For a detailed list of all antibodies used for immunohistochemistry, see Table S1. An Axio Imager M1 (Zeiss) equipped with both ZEN software (blue edition, v2.3) and AxioVision (4.8.2) was used to capture stitched images of whole muscle cross‐sections for all image quantification.