High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain
检测脑中不同亚细胞1型大麻素受体结构域的高分辨率免疫电镜技术   

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Addiction Biology
Nov 2017

 

Abstract

Activation of type 1 cannabinoid (CB1) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (i.e., CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB1 receptors in the brain. In situ hybridization and immunohistochemical techniques have been crucial for defining the CB1 receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB1 receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (e.g., glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB1 receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB1 receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB1 receptors and protein markers of specific brain cells or synapses (e.g., GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB1 receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.

Keywords: Endocannabinoid system (内源性大麻素系统), Cannabinoid receptors (大麻素受体), Receptor localization (受体定位), Immunohistochemistry (免疫组化), Pre-embedding immunogold (预包埋免疫金), Pre-embedding immunoperoxidase (预包埋免疫过氧化物酶), Post-embedding immunogold (包埋后免疫金), Electron microscopy (电子显微镜)

Background

The endocannabinoid system (eCBs) is widely distributed in the nervous system, and, through the activation of CB1 receptors, plays an important role in normal brain function (Herkenham et al., 1990; Tsou et al., 1998; Kano et al., 2009; Castillo, 2012; Katona and Freund, 2012; Lutz et al., 2015; Pertwee, 2015; Lu and Mackie, 2016; Busquets-Garcia et al., 2018).

Pre- and post-embedding immunocytochemical techniques for electron microscopy are valuable methods that can provide precise CB1 receptor localization in brain and peripheral tissues. The pre-embedding immunogold labeling method has been successfully employed in our laboratory for the localization of various receptors, ion channels and enzymes in the mammalian brain, and has served to reveal a unique and interesting presence of CB1 receptors in mitochondria (Bénard et al., 2012; Hebert-Chatelain et al., 2014 and 2016; Mendizabal-Zubiaga et al., 2016; Gutiérrez-Rodríguez et al., 2018). The advantages are reflected by its ease of applicability and relatively inexpensive tissue preparation equipment and relatively good ultrastructural preservation. In addition, it offers the ability to perform correlative light microscopy on stained sections in order to reveal general patterns of CB1 receptor labeling prior to examination with the electron microscope. Success of the pre-embedding detection method has been improved by the development of ultra-small gold secondary conjugates (Nanogold®) in combination with gold particle enlarging chemistry, making it possible for probes to detect deeper into tissues, thus reducing the inherent limitation of antibody penetration and molecule detection while enhancing the resolution of receptor localization. In combination with immunoperoxidase and 3,3’-diaminobenzidine (DAB) reaction product immunochemistry, pre-embedding procedures can reveal protein co-localizations with cell specificity and at high resolution.

With post-embedding immunogold labeling methods, the epitopes are theoretically exposed at the section surface, diffusion of immunoreagents is avoided, is quantitative, allows simultaneous labeling by using different gold particle sizes (Hermida et al., 2010, Hunt et al., 2013) and the labeling is reliable on serial ultrathin sections (Hermida et al., 2006). However, the sensitivity is only moderate, not all antibodies work successfully in the resin-embedded tissue conditions required for the technique, and membrane and structural protein visibility is typically compromised since post-fixation with osmium tetroxide is avoided due to its destructive effects on antigens. Last, but not least, the equipment required for post-embedding labeling procedures can be unaffordable.

Materials and Reagents

  1. Glass vials (Thermo Fisher Scientific, catalog number: C4010-LV1)
  2. Glass slides (Sigma-Aldrich, catalog number: S8902)
  3. Aluminum foil (Sigma-Aldrich, catalog number: 326852)
  4. Syringes (Proquinorte)
  5. Nickel mesh grids: grids for pre-embedding electron microscopy (Electron Microscopy Sciences, catalog number: G-150 Ni), storage temperature: RT
  6. Nickel single slot formvar coated grids (aperture grids: grids for post-embedding electron microscopy) (Electron Microscopy Sciences, catalog number: FFGA1000-Ni-50), storage temperature: RT
  7. Antibodies
    Antibody
    Manufacturer; species; catalog number; RRID
    Anti-cannabinoid receptor type-1 (CB1)
    Frontier Institute Co., ltd; goat polyclonal; CB1-Go-Af450; AB_2571592
    Anti-cannabinoid receptor type-1 (CB1)
    Frontier Institute Co., ltd; guinea pig polyclonal; CB1-GP-Af530; AB_2571593
    Anti-glial fibrillary acidic protein (GFAP)
    Sigma-Aldrich; mouse monoclonal; G3893; AB_257130
    Anti-gephyrin
    Synaptic Systems; mouse monoclonal; 147021; AB_2232546
    Anti-A522 (EAAT1 [GLAST]) Prof. Niels Christian Danbolt University of Oslo; rabbit polyclonal; Ab#314; AB_2314561
    Anti-metabotropic glutamate receptor 2/3 (mGluR2/3)
    Chemicon (Millipore); rabbit polyclonal; AB1553, AB_11212089
    Biotinylated anti-mouse secondary antibody
    Vector Labs; BA-2000; AB_2313581
    Biotinylated anti-rabbit secondary antibody
    Vector Labs; BA-1000; AB_2313606
    1.4 nm gold-conjugated anti-guinea pig IgG (Fab’ fragment) secondary antibody
    Nanoprobes; goat; #2055
    1.4 nm gold-conjugated anti-goat IgG (Fab’ fragment) antibody
    Nanoprobes; rabbit; #2004
    Colloidal gold-18 nm anti-goat IgG antibody
    Jackson Immunoresearch; donkey; 705-215-147
    F(ab’)2 anti-rabbit IgG fragments conjugated to 10 nm colloidal gold particles
    British Biocell International; goat; GFAR10

  8. Lowicryl HM20 kit (Sigma-Aldrich, catalog number: 15924), storage temperature: RT
  9. VECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard) (Vector Laboratories, catalog number: PK-6100), storage temperature:
    4 °C
  10. HQ Silver: silver enhancement kit for EM (Nanoprobes, catalog number: 2012), storage temperature: -20 °C
  11. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906), storage temperature: 4 °C
  12. 3,3′-diaminobenzidine tetrahydrochloride hydrate (DAB): C12H14N4•4HCl•xH2O (Sigma-Aldrich, catalog number: D5637), storage temperature: -20 °C
  13. Ethanol absolute: CH3CH2OH (PanReac AppliChem, catalog number: A1613), storage temperature: RT
  14. Glycerol: HOCH2CH(OH)CH2OH (Sigma-Aldrich, catalog number: G9012), storage temperature: RT
  15. Glycine: NH2CH2COOH (Sigma-Aldrich, catalog number: G8898), storage temperature: RT
  16. Ketamine hydrochloride/xylazine hydrochloride solution for anesthesia (Sigma-Aldrich, catalog number: K4138), storage temperature: 4 °C
  17. Methanol: CH3OH (Sigma-Aldrich, catalog number: 322415), storage temperature: RT
  18. Propane: CH3CH2CH3 (Sigma-Aldrich, catalog number: 536172), storage temperature: RT
  19. (R)-(+)-propylene oxide: C3H6O (Sigma-Aldrich, catalog number: 540048), storage temperature: RT
  20. Saponin (Sigma-Aldrich, catalog number: 84510), storage temperature: RT
  21. Sodium azide (NaN3) (PanReac AppliChem, catalog number: 122712.1609), storage temperature: RT
  22. Sodium borohydride: NaBH4 (Sigma-Aldrich, catalog number: 71320), storage temperature: RT
  23. Osmium tetroxide aqueous solution (4%) (Electron Microscopy Sciences, catalog number: 19150), storage temperature: RT
  24. Di-Sodium hydrogen phosphate (Na2HPO4) (Merck, catalog number: 1.06586.1000), storage temperature: room temperature (RT) (used in Recipes 1-3)
  25. Epoxy embedding medium, hardener DDSA: C16H26O3 (Sigma-Aldrich, catalog number: 45346), storage temperature: RT (used in Recipe 6)
  26. Epoxy embedding medium, hardener MNA: C10H10O3 (Sigma-Aldrich, catalog number: 45347), storage temperature: RT (used in Recipe 6)
  27. Epoxy embedding medium: EponTM 812 substitute (Sigma-Aldrich, catalog number: 45345), storage temperature: RT (used in Recipe 6)
  28. Glutaraldehyde (25%) in aqueous solution for synthesis: C5H8O2 (Merck, catalog number: 8.20603.1000), storage temperature: 4 °C (used in Recipe 1)
  29. Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: H1758), storage temperature: RT (used in Recipe 2)
  30. Lead(II) nitrate: (PbNO3)2 (PanReac AppliChem, catalog number: 131473), storage temperature: RT (used in Recipe 8)
  31. N-benzyldimethylamine: C9H13N (Sigma-Aldrich, catalog number: 185582), storage temperature: RT (used in Recipe 6)
  32. Paraformaldehyde: (CH2O)n (Merck, catalog number: 1.04005.1000), storage temperature: RT (used in Recipe 1)
  33. Picric acid solution (1.3%) dissolved in H2O (saturated): (O2N)3C6H2OH (Sigma-Aldrich, catalog number: P6744), storage temperature: RT (used in Recipe 1)
  34. Poly(ethylene glycol) (PEG): C2nH4nH2On+1 (Sigma-Aldrich, catalog number: 202444), storage temperature: RT
  35. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541), storage temperature: RT (used in Recipe 2)
  36. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: 229806), storage temperature: RT (used in Recipe 2)
  37. Sodium chloride (NaCl) (PanReac AppliChem, catalog number: 131659.1211), storage temperature: RT (used in Recipes 2-4)
  38. Sodium hydroxide pellets (PanReac AppliChem, catalog number: 131687.1211), storage temperature: RT (used in Recipe 8)
  39. Sodium phosphate monobasic monohydrate (NaH2PO4•H2O) (PanReac AppliChem, catalog number: 131965.1211), storage temperature: RT (used in Recipes 1-3)
  40. Tri-sodium citrate 2-hydrate: Na3C6H5O7•2H2O (Merck, catalog number: 6448), storage temperature: 4 °C (used in Recipe 8)
  41. Triton X-100 (Sigma-Aldrich, catalog number: X100), storage temperature: RT (used in Recipe 5)
  42. Trizma® base: NH2C(CH2OH)3 (Sigma-Aldrich, catalog number: T1503), storage temperature: RT (used in Recipes 4 and 5)
  43. Trizma® hydrochloride: NH2C(CH2OH)3•HCl (Sigma-Aldrich, catalog number: T3253), storage temperature: RT (used in Recipe 4)
  44. Uranyl acetate: UO2(CH3COO)2 (Electron Microscopy Sciences, catalog number: 22400), storage temperature: RT (used in Recipe 7)
  45. Fixative solution (see Recipes)
  46. Phosphate buffered saline (PBS 1x) (see Recipes)
  47. 0.1 M phosphate buffer (0.1 M PB) (see Recipes)
  48. Tris-hydrogen chloride buffered saline (TBS 1x) (see Recipes)
  49. Tris-buffered saline with Triton X-100 (TBST) (see Recipes)
  50. Epon resin (see Recipes)
  51. Uranyl acetate (2%, aqueous) staining solution (see Recipes)
  52. Reynold’s lead citrate staining solution (see Recipes)

Equipment

  1. Erlenmeyer flasks (Sigma-Aldrich, catalog number: Z567868)
  2. Shaker (Heidolph, Duomax 1030)
  3. Oven (Grupo Selecta, Dryterm)
  4. Vibratome (Leica Biosystems, model: Leica VT1000 S)
  5. Super platinum knife (Gillette)
  6. Ultramicrotome (RMC Products, model: PowerTome XL)
  7. Histo 6 mm diamond knife 45° (Diatome)
  8. Ultra 2.4 mm diamond knife 45° (Diatome)
  9. Transmission electron microscope (Philips, model: EM208S)
  10. Digital Morada camera (Olympus SIS Morada camera)
  11. Cryofixation unit (Reichert-Jung, model: KF 80)
  12. Freeze substitution system (Reichert AFS)

Software

  1. Adobe Photoshop (CS3, Adobe Systems, San Jose, CA, USA)
  2. ImageJ (NIH, USA; RRID:SCR_003070)
  3. GraphPad Prism 5 (GraphPad Software Inc., San Diego, USA; RRID:SCR_002798)

Procedure

  1. PRE-EMBEDDING (Figure 1)
    Preservation of brain tissue
    1. Anesthetize animals (at least n = 3) by intraperitoneal injection of ketamine/xylazine (80/10 mg/kg body weight).
    2. Perfuse animals through the left ventricle with PBS (0.1 M, pH 7.4, 20-25 °C) for ~20 s at RT, followed by ice-cold fixative solution made up of 4% formaldehyde (freshly depolymerized from paraformaldehyde), 0.2% picric acid, and 0.1% glutaraldehyde in PB (0.1 M, pH 7.4). Fixative solution/mouse: 250 ml. Perfusion time 15 min. Fixative solution/rat: 500 ml. Perfusion time: 30 min.
    3. Remove the brain from the skull and post-fix in the fixative solution for ~1 week at 4 °C. Store samples in 0.1 M PB diluted fixative (1:10) containing 0.025% sodium azide at 4 °C until use.


    Figure 1. Timeline of the general steps for pre- and post-embedding immunoelectron microscopy techniques

    Pre-embedding immunogold method for electron microscopy (Figures 1, 2A and 3A)
    1. Cut 50 µm-thick sections on a vibratome and place into 12-well cell culture plates in 0.1 M PB (pH 7.4) at RT. Sections from each brain are in separate plates for storage at 4 °C in 0.1 M PB (pH 7.4) with 0.025% sodium azide. Two or three sections per brain containing the area of interest are selected and placed in a new plate. Total volume per well: 1 ml.
    2. Pre-incubate in blocking solution (1 ml/well) containing 10% BSA, 0.02% saponin and 0.1% sodium azide in TBS 1x (pH 7.4) on a shaker (300 rpm) for 30 min at RT.
    3. Incubate with goat polyclonal anti-CB1 receptor antibody (diluted 1:100, 1 ml/well) diluted in 10% BSA/TBS 1x containing 0.004% saponin and 0.1% sodium azide on a shaker. Place dish on an orbital shaker for two days at 4 °C. 
    4. Wash five times in 1% BSA/TBS (3 x 1 min and 2 x 10 min).
    5. Incubate with 1.4 nm gold-conjugated rabbit anti-goat IgG (Fab' fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA; 1 ml/well) in 1% BSA/TBS with 0.004% saponin on a shaker for 3 h at RT.
    6. Wash three times in 1% BSA/TBS (10 min each). Keep the tissue in 1% BSA/TBS on a shaker overnight at 4 °C.
    7. Post-fix with 1% glutaraldehyde prepared in TBS (1 ml/well) for 10 min at RT.
    8. Wash three times in double distilled water (10 min each). 
    9. Transfer sections to glass test tubes.
    10. Intensify gold particles with the HQ Silver kit (Nanoprobes Inc., Yaphank, NY, USA; 1 ml/tube) in the dark for 12 min. 
    11. Wash three times in double distilled water (1 min each). 
    12. Wash three times in 0.1 M PB (pH 7.4) (10 min each). 
    13. Transfer sections to glass vials (15 ml, 3 x 5 cm).
    14. Osmicate (1% osmium tetroxide in 0.1 M PB, pH 7.4; 1 ml/vial) in the dark for 20 min.
    15. Wash three times in 0.1 M PB (pH 7.4) (10 min each).
    16. Dehydrate in graded ethanols (50%, 70%, 96%; 5 min/each) followed by three times in 100% ethanol (5 min each) (1 ml/vial). 
    17. Replace with propylene oxide (3 x 5 min, 1 ml/vial).
    18. Infiltrate sections with a 1:1 mixture of propylene oxide and Epon resin 812 (1 ml/vial) on a shaker overnight at RT. 
    19. Mix with pure Epon resin 812 (1 ml/vial) for > 2 h at RT.
    20. Place sections between two glass slides and wrap slides with aluminum foil.
    21. Polymerize resin-embedded sections at 60 °C for 2 days.
    22. Cut 1 μm semi-thin sections on ultramicrotome.
    23. Trim block and cut 60 nm ultra-thin sections with a diamond knife and collect on nickel mesh grids. 
    24. Stain sections with 2.5% lead citrate (1 drop/grid) for 20 min at RT. 
    25. Wash three times in double distilled water (1 drop/grid) (10 min each).
    26. Examine under a Philips EM208S transmission electron microscope. 
    27. Photograph sections using a digital Morada camera (Olympus).

    Double pre-embedding immunogold and immunoperoxidase method (Figures 1, 2B and 3B-3D)
    1. Cut 50 µm-thick sections on vibratome and collect in 12-well cell culture plates in 0.1 M PB (pH 7.4) at RT. Sections from each brain are in separate plates for storage at 4 °C in 0.1 M PB (pH 7.4) with 0.025% sodium azide. Two or three sections per brain containing the area of interest are selected and placed in a new plate. Total volume per well: 1 ml.
    2. Pre-incubate in blocking solution (1 ml/well) containing 10% BSA, 0.02% saponin and 0.1% sodium azide in TBS 1x, pH 7.4 on a shaker (300 rpm) for 30 min at RT. 
    3. Incubate sections with goat polyclonal anti-CB1 receptor antibody or guinea pig polyclonal anti-CB1 receptor antibody (diluted 1:100, 1 ml/well) in combination with either a mouse monoclonal anti-GFAP antibody (1:1,000), rabbit polyclonal anti-GLAST antibody (0.3 µg/ml), or mouse monoclonal anti-gephyrin antibody (1:250) prepared in 10% BSA/TBS 1x containing 0.004% saponin and 0.1% sodium azide. Place dish on an orbital shaker for 2 days at 4 °C.
      Note: The guinea pig polyclonal anti-CB1 antibody is used for double immunolabeling with the rabbit polyclonal anti-GLAST antibody.
    4. Wash five times in 1% BSA/TBS (3 x 1 min and 2 x 10 min).
    5. Incubate with corresponding biotinylated secondary antibody (1:200) and 1.4 nm gold-conjugated secondary rabbit anti-goat IgG (Fab’ fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA) or 1.4 nm gold-conjugated secondary goat anti-guinea pig IgG (Fab’ fragment, 1:100, Nanoprobes Inc., Yaphank, NY, USA) diluted in 1% BSA/TBS with 0.004% saponin on a shaker for 4 h at RT.
    6. Wash three times in 1% BSA/TBS (10 min each) on a shaker at RT. 
    7. Incubate in avidin-biotin-peroxidase complex (ABC) (1:50) prepared in washing solution (1 ml/well) for 1.5 h at RT. 
    8. Wash three times in 1% BSA/TBS (10 min each). Keep tissue in 1% BSA/TBS on a shaker overnight at 4 °C.
    9. Post-fix with 1% glutaraldehyde in TBS (1 ml/well) for 10 min at RT. 
    10. Wash three times in double distilled water (10 min each). 
    11. Sections are transferred to test tubes.
    12. Intensify gold particles with the HQ Silver kit (Nanoprobes Inc., Yaphank, NY, USA; 1 ml/tube) in the dark for 12 min. 
    13. Wash three times in double distilled water (1 min each). 
    14. Wash three times in 0.1 M PB (pH 7.4) (10 min each). 
    15. Sections are transferred to glass vials (15 ml, 3 x 5 cm).
    16. Incubate in 0.05% DAB and 0.01% hydrogen peroxide prepared in 0.1 M PB (1 ml/vial) for 3 min at RT. 
    17. Wash three times in 0.1 M PB (pH 7.4) (10 min each). 
    18. Osmicate samples (1% osmium tetroxide in 0.1 M PB, pH 7.4; 1 ml/vial) in the dark for 20 min.
    19. Wash three times in 0.1 M PB (pH 7.4) (10 min each).
    20. Dehydrate in graded ethanols (50%, 70%, 96%; 5 min/each) followed by three times in 100% ethanol (5 min each) (1 ml/vial). 
    21. Replace with propylene oxide (3 x 5 min, 1 ml/vial).
    22. Infiltrate sections with a 1:1 mixture of propylene oxide and Epon resin 812 (1 ml/vial) on a shaker overnight at RT. 
    23. Mix with pure Epon resin 812 (1 ml/vial) for > 2 h at RT.
    24. Place sections between two glass slides and wrap slides in aluminum foil.
    25. Polymerize resin-embedded sections in an oven at 60 °C for 2 days. 
    26. Cut 1 μm semi-thin sections on ultramicrotome.
    27. Trim block and cut 60 nm ultra-thin sections with a diamond knife and collect on nickel mesh grids. 
    28. Stain sections with 2.5% lead citrate (1 drop/grid) for 20 min at RT. 
    29. Wash three times in double distilled water (1 drop/grid) (10 min each).
    30. Examine under a Philips EM208S transmission electron microscope. 
    31. Photograph sections using a digital Morada camera (Olympus).

  2. POST-EMBEDDING (Figure 1)
    Single/double labeling with post-embedding immunogold method (Figures 1, 2C and 3E-3F)
    1. Preservation of brain tissue. For optimal ultrastructure, anesthetize and perfuse the animals as above. 
    2. Remove the brain from the skull and post-fix in the fixative solution for ~1 week at 4 °C. Then, store the brains in 0.1 M PB diluted fixative (1:10) plus 0.025% sodium azide at 4 °C and rinse in PB until next step. 
    3. Specimen trimming (cut small rectangular pieces of 0.5 x 0.5 x 1 mm from the region of interest).
    4. Cryoprotect in glycerol (10%, 20%, and 30% in PB) and rapidly freeze in liquid propane using a cryofixation unit (KF80; Reichert, Vienna, Austria). 
    5. Freeze-substitute with methanol and 0.5% uranyl acetate.
    6. Embed in Lowicryl HM20 (Lowi, Waldkraiburg, Germany).

    Immunogold labeling procedure
    1. Cut 1 μm semi-thin sections on ultramicrotome.
    2. Collect 70 nm ultra-thin sections on nickel single slot formvar coated grids.
    3. Wash specimens with TBST containing 0.1% NaBH4 and 50 mM glycine (10 μl drop/grid) for 10 min. 
    4. Rinse 3 times in TBST (10 μl drop/grid) (1 min each).
    5. Pre-incubate in blocking solution (10 μl drop/grid) containing 10% BSA in TBST for 10 min.
    6. Incubate (10 μl drop/grid) with anti-CB1 receptor (1:50) or anti-mGluR2/3 (1:50) antibodies or with a mixture of them prepared in 2% BSA/TBST, overnight at 4 °C. 
    7. Wash three times in TBST (10 μl drop/grid) (10 min each).
    8. Pre-incubate in blocking solution (10 μl drop/grid) containing 10% BSA and 0.5% PEG in TBST for 10 min. 
    9. Incubate (10 μl drop/grid) with secondary antibodies coupled to different gold particle size: donkey 18 nm colloidal gold conjugated affinity purified anti-goat IgG (for CB1) and goat F(ab’)2 anti-rabbit IgG coupled to 10 nm colloidal gold particles (for mGluR2/3), diluted 1:20 in 2% BSA/TBST for 2 h at RT.
    10. Wash three times in double-distilled water (10 μl drop/grid) (10 min each).
    11. Counterstain with 2% uranyl acetate (20 min) and 2.5% lead citrate (2 min) (10 μl drop/grid).
    12. Wash three times in double distilled water (10 μl drop/grid) (10 min each).
    13. Examine under a Philips EM208S transmission electron microscope. 
    14. Photograph specimens using a digital Morada camera (Olympus).


    Figure 2. Schematics illustrating the three immunolabeling methods for high-resolution electron microscopy


    Figure 3. CB1 receptor immunolocalization in different subcellular compartments of the rodent brain. Single pre-embedding immunogold (A) and double pre-embedding immunogold and immunoperoxidase methods (B-D). A. CB1 receptor labeling (arrows) at a presynaptic GABAergic terminal (ter, yellow) adjacent to a dendrite (den, purple). CB1 receptor particle is localized to a presynaptic glutamatergic terminal (ter, pink) associated with a spine (sp, purple). Mitochondria (m, red) exhibit CB1 receptor immunolabeling in both glutamatergic (ter, pink) and GABAergic (ter, yellow) presynaptic terminals (CA1 stratum radiatum, adult mouse hippocampus). B. CB1 receptor labeling (arrows) at a presynaptic GABAergic terminal (ter, yellow), glutamatergic terminals (ter, purple) and in one astrocyte branch (white arrowhead; as, green) in the mouse piriform cortex. Astrocytes are labeled with anti-GLAST/immunoperoxidase/DAB method (black precipitate in as). C. CB1 receptor labeling (arrows) at a presynaptic GABAergic terminal (ter, yellow) adjacent to a dendrite (den, purple) and in one astrocyte process (white arrowhead; as, green) in the molecular layer of the mouse dentate gyrus. Astrocytes are labeled with anti-GFAP/immunoperoxidase/DAB method (black precipitate in as). D. CB1 receptor labeling (arrows) at a presynaptic terminal (ter, yellow) combined with anti-gephyrin/immunoperoxidase/DAB method (black precipitate in den, purple) to positively identify the inhibitory postsynaptic membrane of a GABAergic synapse. White arrowhead: CB1 receptor labeling at a thin astrocytic process filling the intercellular space (rat prelimbic cortex). E-F. Double post-embedding immunogold method revealing the localization of presynaptic CB1 receptors (ter; 18 nm-diameter gold particles) and postsynaptic mGluR2/3 (den, sp; 10 nm-diameter gold particles) at inhibitory (ter, yellow) and excitatory (ter, pink) synapses in the mouse dentate molecular layer. Scale bars= 500 nm.

Data analysis

  1. Semi-quantification of CB1 receptor immunolabeling in pre-embedding method
    With the aim of maximizing the standard conditions, the pre-embedding immunogold method is applied simultaneously to all the sections obtained from the animals under study (at least n = 3). Three replicated experiments are done for each animal.
      Immunogold-labeled resin-embedded vibratome sections are first visualized under the light microscope in order to select portions of the region of interest (i.e., CA1 hippocampus, dentate molecular layer, prelimbic cortex or piriform cortex) with reproducible CB1 receptor immunolabeling. Then, semi-thin sections from resin-embedded tissue are cut and the first five ultra-thin sections are collected onto two grids. To further standardize the conditions between the different animals, only the first 1.5 µm from each specimen surface is collected and randomly photographed. Sampling is always performed carefully and in the same way for all the animals studied. To avoid bias, investigators remained blind when taking and analyzing the electron micrographs.
      The excitatory and inhibitory synapses are identified by their ultrastructural features; excitatory synapses are asymmetrical with postsynaptic densities and presynaptic axon terminals containing abundant, clear and spherical synaptic vesicles. Inhibitory synapses are symmetrical with slender postsynaptic membranes and axon terminals containing pleomorphic synaptic vesicles. Because of the lack of postsynaptic membrane density, the inhibitory nature of the synapse might be misleading unless serial sections were done. An alternative to circumvent this is to use an antibody against gephyrin, a postsynaptic anchor protein marker of inhibitory synapses which can be used to unequivocally identify inhibitory synapses. CB1 receptors in astrocytes are assessed in astrocytic processes containing GFAP or GLAST DAB immunodeposits.
      The proportion of the CB1 receptor labeling on different compartments identified as described above is then tabulated. Positive labeling is considered when at least one CB1 receptor immunoparticle is within ~30 nm of the membrane of the specific compartment under study, and ≥ 80 nm from other membranes in the case of mitochondrial labeling. Metal particles are then counted and CB1 receptor density (particles/µm membrane) in the positive compartments is determined with ImageJ software by measuring their membrane length. We also estimate the proportion of CB1 receptor immunoparticles in different profiles versus the total CB1 receptor expression. This gives information about the CB1 receptor distribution throughout different compartments of a particular brain region (excitatory and inhibitory synapses, astrocytes, mitochondria, other cellular compartments). As for astrocytes, the distance from astrocytic CB1 receptor immunoparticles to the nearest synapse is also calculated to determine how the receptors are distributed in the context of the tripartite synapse. To do this, the nearby synapses surrounding the CB1 receptor positive astrocytic elements are identified, distances measured (ImageJ software), the nearest synapse to the astrocytic immunoparticle selected, and data from all the nearest synapses tabulated and analyzed.
      All values are given as mean ± S.E.M. using a statistical software package (GraphPad Prism 5, GraphPad Software Inc., San Diego, USA). The normality test (Kolmogorov-Smirnov normality test) is always applied before running statistical tests. Data are analyzed using parametric or non-parametric two-tailed Student’s t-test or one-way ANOVA with subsequent post-hoc analysis (Bonferroni post-test).

  2. Quantification of CB1 receptor immunolabeling in post-embedding method
    Electron micrographs of identified excitatory and inhibitory synaptic terminals are randomly obtained from a particular brain region (e.g., molecular layer of the dentate gyrus). For analysis, 50 synapses containing the criteria of intact plasma membranes, synaptic vesicles and prominent synaptic clefts are selected from each animal studied. To avoid bias, investigators remained blind when taking and analyzing the electron micrographs. The percentage of CB1 receptor positive excitatory and/or inhibitory terminals and the percentage of mGluR2/3 labeled postsynaptic dendrites receiving CB1 positive presynaptic terminals are analyzed using a statistical software package (GraphPad Prism 5, GraphPad Software Inc., San Diego, CA, USA). Labeling is considered positive as described in the pre-embedding techniques (e.g., membrane proximity). Data are shown as mean ± S.E.M. CB1 receptor density (particles/µm membrane) in positive terminals is calculated as explained before. In addition, with the aim of determining the precise sub-synaptic distribution of mGluR2/3 (or any other receptor) in postsynaptic elements relative to the presynaptic release sites, the distribution of gold particles frequency is measured. Also, the sub-synaptic CB1 receptor distribution in presynaptic boutons can be determined. In this situation, the synaptic or peri/extrasynaptic localization of the gold particle is defined with respect to its allocation in 60 nm-wide segments obtained from the edge of both the postsynaptic density and the presynaptic active zone (localization at the edge = 0). Statistical analysis is performed as described in the pre-embedding method section.

Recipes

  1. Fixative solution
    Heat 600 ml of distilled water in a microwave to 60 °C
    Add 11.5 g of Na2HPO4 (anhydrous) (MW: 141.96)
    Add 40 g of paraformaldehyde (MW: 30.03) and shake at 60 °C. It could take 30 min to dissolve
    Add 2.62 g NaH2PO4•H2O (MW: 137.99)
    Filter the solution into an Erlenmeyer flask and add 2 ml of saturated picric acid (MW: 229.11)
    Make up to 1 L with distilled water
    Cool and store at 4 °C until used
    Just before perfusion, add 4 ml of glutaraldehyde (25%)
  2. Phosphate buffered saline (PBS 1x) (0.1 M, pH 7.4)
    For 1 L of PBS 1x, prepare as follows:
    Start with 800 ml of distilled water:
    Add 8 g of NaCl (MW: 58.44)
    Add 0.2 g of KCl (MW: 74.55)
    Add 1.44 g of Na2HPO4 (MW: 141.96)
    Add 0.24 g of KH2PO4 (MW: 136.09)
    Adjust the pH to 7.4 with HCl (MW: 36.46)
    Add distilled water to a total volume of 1 L
  3. 0.1 M phosphate buffer (0.1 M PB) pH = 7.4
    Stock solution: 0.2 M PB, pH = 7.4
    For 1 L of 0.2 M PB, prepare as follows:
    Add 5.24 g of NaH2PO4•H2O (MW: 138)
    Add 23.0 g of Na2HPO4 (MW: 141.96)
    Distilled water, make up the solution to 1 L
    To prepare 0.1 M PB, dilute 1:1 the stock solution 0.2 M PB in distilled water
  4. Tris-hydrogen chloride buffered saline: 3 M NaCl + 1 M Tris-HCl, pH = 7.4; (TBS 1x)
    Stock solution: 0.3 M NaCl + 0.1 M Tris-HCl, pH = 7.4 (10x TBS)
    For 1 L of 10x TBS, prepare as follows:
    Add 175 g of NaCl (MW: 58.44)
    Add 19.4 g of Trizma base (MW: 121.14)
    Add 132.2 g of Trizma-HCl (MW: 157.60)
    Make up solution to 1 L with distilled water
    To prepare 1 L of TBS 1x, dilute 1:9 the TBS 10x stock solution in distilled water
  5. Tris-buffered saline with Triton X-100 (TBST)
    Take 450 ml of distilled water
    Add 3.03 g of Trizma base (MW: 121.14)
    Add 4.5 g of NaCl (MW: 58.44)
    Adjust the pH to 7.4 and make the solution up to 500 ml with distilled water
    Add 0.5 ml of Triton X-100 (MW: 646.86)
  6. Epon resin
    Add 81.3 g of EPON 812 (MW: 178.18)
    Add 53.0 g of EPON HÄRTER DDSA (MW: 266.38)
    Add 35.7 g of MNA (MW: 178.18)
    Add 2.24 ml of N-Benzyldimethylamine Fluka 13370 (MW: 135.21)
    Mix for at least 2 h before storing at -20 °C in syringes
  7. Uranyl acetate (2%, aqueous) staining solution
    Prepare 0.04 g uranyl acetate [UO2(CH3COO)2 (MW: 388.11)] in 2 ml of distilled water, Centrifuge at 11,600 x g for 5 min and use supernatant
  8. Reynold’s lead citrate staining solution
    Add 2.66 g of lead nitrate [Pb(NO3)2, MW 331.2)] and 3.52 g of Tri-sodium citrate dihydrate (Na3C6H5O7•2H2O, MW: 294.10) in 84 ml of double distilled water (it is normal for the solution to become cloudy when sodium citrate is added)
    Prepare 0.8 g of 1 N NaOH (MW: 39.99) in 20 ml double distilled water
    Add 16 ml of NaOH solution to the lead citrate solution (solution becomes clear when NaOH is added)
    Filter the solution to remove any undissolved material

Acknowledgments

This work was supported by The Basque Government (BCG IT764-13); MINECO/FEDER, UE (SAF2015-65034-R); Red de Trastornos Adictivos, Instituto de Salud Carlos III (ISC-III) and European Regional Development Funds-European Union (ERDF-EU) (RD16/0017/0012). We would like to thank Prof. Niels Christian Danbolt for providing us with the rabbit polyclonal anti-A522 EAAT1 [GLAST] antibody and Prof. Mahmood Amiry-Moghaddam and Bjørg Riber for the Lowicryl HM20 embedding (Division of Anatomy, Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Norway).

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

The protocols for animal care and use were approved by the Committee of Ethics for Animal Welfare of the University of the Basque Country (CEEA/M20/2016/073; CEIAB/2016/074) and were in accordance to the European Communities Council Directive of 22nd September 2010 (2010/63/EU) and Spanish regulations (Real Decreto 53/2013, BOE 08-02-2013).

References

  1. Bénard, G., Massa, F., Puente, N., Lourenço, J., Bellocchio, L., Soria-Gómez, E., Matias, I., Delamarre, A., Metna-Laurent, M., Cannich, A., Hebert-Chatelain, E., Mulle, C., Ortega-Gutiérrez, S., Martín-Fontecha, M., Klugmann, M., Guggenhuber, S., Lutz, B., Gertsch, J., Chaouloff, F., López-Rodríguez, M. L., Grandes, P., Rossignol, R. and Marsicano, G. (2012). Mitochondrial CB1 receptors regulate neuronal energy metabolism. Nat Neurosci 15: 558-564.
  2. Busquets-Garcia, A., Bains, J. and Marsicano, G. (2018). CB1 receptor signaling in the brain: extracting specificity from ubiquity. Neuropsychopharmacology 43(1): 4-20.
  3. Castillo, P. E. (2012). Presynaptic LTP and LTD of excitatory and inhibitory synapses. Cold Spring Harb Perspect Biol 4(2): a005728-a005728.
  4. Gutiérrez-Rodríguez, A., Bonilla-Del Rio, I., Puente, N., Gomez-Urquijo, S. M., Fontaine, C. J., Egana-Huguet, J., Elezgarai, I., Ruehle, S., Lutz, B., Robin, L. M., Soria-Gomez, E., Bellocchio, L., Padwal, J. D., van der Stelt, M., Mendizabal-Zubiaga, J., Reguero, L., Ramos, A., Gerrikagoitia, I., Marsicano, G. and Grandes, P. (2018). Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus. Glia 66(7): 1417-1431.
  5. Hebert-Chatelain, E., Desprez, T., Serrat, R., Bellocchio, L., Soria-Gomez, E., Busquets-Garcia, A., Pagano Zottola, A. C., Delamarre, A., Cannich, A., Vincent, P., Varilh, M., Robin, L. M., Terral, G., Garcia-Fernandez, M. D., Colavita, M., Mazier, W., Drago, F., Puente, N., Reguero, L., Elezgarai, I., Dupuy, J. W., Cota, D., Lopez-Rodriguez, M. L., Barreda-Gomez, G., Massa, F., Grandes, P., Benard, G. and Marsicano, G. (2016). A cannabinoid link between mitochondria and memory. Nature 539(7630): 555-559.
  6. Hebert-Chatelain, E., Reguero, L., Puente, N., Lutz, B., Chaouloff, F., Rossignol, R., Piazza, P. V., Benard, G., Grandes, P. and Marsicano, G. (2014). Cannabinoid control of brain bioenergetics: Exploring the subcellular localization of the CB1 receptor. Mol Metab 3(4): 495-504.
  7. Herkenham, M., Lynn, A. B., Little, M. D., Johnson, M. R., Melvin, L. S., de Costa, B. R. and Rice, K. C. (1990). Cannabinoid receptor localization in brain. Proc Natl Acad Sci U S A 87(5): 1932-1936.
  8. Hermida, D., Elezgarai, I., Puente, N., Alonso, V., Anabitarte, N., Bilbao, A., Donate-Oliver, F. and Grandes, P. (2006). Developmental increase in postsynaptic alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid receptor compartmentalization at the calyx of Held synapse. J Comp Neurol 495(5): 624-634.
  9. Hermida, D., Mateos, J. M., Elezgarai, I., Puente, N., Bilbao, A., Bueno-Lopez, J. L., Streit, P. and Grandes, P. (2010). Spatial compartmentalization of AMPA glutamate receptor subunits at the calyx of Held synapse. J Comp Neurol 518(2): 163-174.
  10. Hunt, D. L., Puente, N., Grandes, P. and Castillo, P. E. (2013). Bidirectional NMDA receptor plasticity controls CA3 output and heterosynaptic metaplasticity. Nat Neurosci 16(8): 1049-1059.
  11. Kano, M., Ohno-Shosaku, T., Hashimotodani, Y., Uchigashima, M. and Watanabe, M. (2009). Endocannabinoid-mediated control of synaptic transmission. Physiol Rev 89(1): 309-380.
  12. Katona, I. and Freund, T. F. (2012). Multiple functions of endocannabinoid signaling in the brain. Annu Rev Neurosci 35: 529-558.
  13. Lu, H. C. and Mackie, K. (2016). An introduction to the endogenous cannabinoid system. Biol Psychiatry 79(7): 516-525.
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  15. Mendizabal-Zubiaga, J., Melser, S., Benard, G., Ramos, A., Reguero, L., Arrabal, S., Elezgarai, I., Gerrikagoitia, I., Suarez, J., Rodriguez De Fonseca, F., Puente, N., Marsicano, G. and Grandes, P. (2016). Cannabinoid CB1 receptors are localized in striated muscle mitochondria and regulate mitochondrial respiration. Front Physiol 7: 476.
  16. Pertwee, R. G. (2015). Endocannabinoids and their pharmacological actions. In: Handbook of Experimental Pharmacology. pp 1-37.
  17. Tsou, K., Brown, S., Sanudo-Pena, M. C., Mackie, K. and Walker, J. M. (1998). Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central nervous system. Neuroscience 83(2): 393-411.

简介

通过内源性,外源性(大麻衍生物)或合成大麻素(即,CP 55.940,Win-2)激活1型大麻素(CB 1 )受体具有多种多样性由于脑中存在CB 1 受体而导致的行为影响。 原位杂交和免疫组织化学技术对于定义CB 1 受体表达和细胞水平定位至关重要。然而,需要更先进的方法来揭示大脑中CB 1 受体的精确形貌,特别是在其他细胞类型和受体表达低的细胞器等未预料到的位点(例如,谷氨酸能神经元,星形胶质细胞,线粒体)。高分辨率免疫电子显微镜为脑中CB 1 受体的亚细胞定位提供了更精确的检测方法。在这里,我们描述了一种基于使用特定CB 1 受体抗体和银强化的1.4 nm金标记Fab'片段的电子显微镜预嵌入免疫金方法,以及组合预嵌入免疫金和免疫过氧化物酶方法,使用生物素化的二抗和抗生物素蛋白 - 生物素 - 过氧化物酶复合物同时定位CB 1 受体和特定脑细胞或突触的蛋白标记( eg , GFAP,GLAST,IBA-1,PSD-95,gephyrin)。此外,还描述了嵌入后免疫金方法并将其与预嵌入标记程序进行比较。这些方法提供了一种相对容易和有用的方法,用于揭示脑中谷氨酸能突触,星形胶质细胞,神经元和星形细胞线粒体中少量CB 1 受体的亚细胞定位。

【背景】 内源性大麻素系统(eCBs)广泛分布于神经系统,通过激活CB 1 受体,在正常脑功能中起重要作用(Herkenham et al。,1990; Tsou et al。,1998; Kano et al。,2009; Castillo,2012; Katona and Freund,2012; Lutz et al。,2015; Pertwee,2015; Lu和Mackie,2016; Busquets-Garcia et al。,2018)。

用于电子显微镜的嵌入前和嵌入后免疫细胞化学技术是可以在脑和外周组织中提供精确的CB 1 受体定位的有价值的方法。预包埋免疫金标记方法已成功应用于我们的实验室,用于定位哺乳动物大脑中的各种受体,离子通道和酶,并有助于揭示CB 1 的独特和有趣的存在。线粒体中的受体(Bénard et al。,2012; Hebert-Chatelain et al。,2014和2016; Mendizabal-Zubiaga et al。, 2016;Gutiérrez-Rodríguez et al。,2018)。其优点体现在其易于施用和相对便宜的组织制备设备和相对良好的超微结构保存。此外,它提供了在染色切片上进行相关光学显微镜检查的能力,以便在用电子显微镜检查之前揭示CB 1 受体标记的一般模式。通过开发超小金二次结合物(Nanogold ®)与金颗粒扩大化学相结合,改进了预包埋检测方法的成功,使探针可以更深入地检测到组织因此降低了抗体渗透和分子检测的固有限制,同时增强了受体定位的分辨率。结合免疫过氧化物酶和3,3'-二氨基联苯胺(DAB)反应产物免疫化学,预包埋程序可以揭示具有细胞特异性和高分辨率的蛋白质共定位。

使用嵌入后免疫金标记方法,表位在理论上暴露在切片表面,避免了免疫试剂的扩散,是定量的,允许使用不同的金粒径同时标记(Hermida et al。,2010 ,Hunt et al。,2013),标记在连续超薄切片上是可靠的(Hermida et al。,2006)。然而,灵敏度仅适中,并非所有抗体都能在该技术所需的树脂包埋组织条件下成功发挥作用,并且膜和结构蛋白的可见性通常受到损害,因为由于其对抗原的破坏性作用而避免了四氧化锇的后固定。 。最后但并非最不重要的是,后嵌入标签程序所需的设备可能无法承受。

关键字:内源性大麻素系统, 大麻素受体, 受体定位, 免疫组化, 预包埋免疫金, 预包埋免疫过氧化物酶, 包埋后免疫金, 电子显微镜

材料和试剂

  1. 玻璃瓶(Thermo Fisher Scientific,目录号:C4010-LV1)
  2. 玻璃载玻片(Sigma-Aldrich,目录号:S8902)
  3. 铝箔(Sigma-Aldrich,目录号:326852)
  4. 注射器(Proquinorte)
  5. 镍网格栅:用于预嵌入电子显微镜的网格(电子显微镜科学,目录号:G-150 Ni),存储温度:RT
  6. 镍单槽formvar涂层网格(孔径网格:用于嵌入电子显微镜的网格)(电子显微科学,目录号:FFGA1000-Ni-50),存储温度:RT
  7. 抗体


  8. Lowicryl HM20试剂盒(Sigma-Aldrich,目录号:15924),储存温度:RT
  9. VECTASTAIN Elite ABC HRP试剂盒(过氧化物酶,标准品)(Vector Laboratories,目录号:PK-6100),贮存温度:
    4°C
  10. HQ Silver:用于EM的银增强试剂盒(Nanoprobes,目录号:2012),储存温度:-20°C
  11. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906),储存温度:4°C
  12. 3,3'-二氨基联苯胺四盐酸盐水合物(DAB):C 12 H 14 N 4 •4HCl•xH 2 O(Sigma-Aldrich,目录号:D5637),储存温度:-20℃
  13. 乙醇绝对值:CH 3 CH 2 OH(PanReac AppliChem,目录号:A1613),储存温度:RT
  14. 甘油:HOCH 2 CH(OH)CH 2 OH(Sigma-Aldrich,目录号:G9012),储存温度:室温
  15. 甘氨酸:NH 2 CH 2 COOH(Sigma-Aldrich,目录号:G8898),储存温度:RT
  16. 盐酸氯胺酮/盐酸甲苯噻嗪麻醉剂溶液(Sigma-Aldrich,目录号:K4138),储存温度:4°C
  17. 甲醇:CH 3 OH(Sigma-Aldrich,目录号:322415),储存温度:RT
  18. 丙烷:CH 3 CH 2 CH 3 (Sigma-Aldrich,目录号:536172),储存温度:RT
  19. (R) - (+) - 环氧丙烷:C 3 H 6 O(Sigma-Aldrich,目录号:540048),储存温度:RT
  20. 皂苷(Sigma-Aldrich,目录号:84510),储存温度:RT
  21. 叠氮化钠(NaN 3 )(PanReac AppliChem,目录号:122712.1609),储存温度:RT
  22. 硼氢化钠:NaBH 4 (Sigma-Aldrich,目录号:71320),储存温度:RT
  23. 四氧化锇水溶液(4%)(Electron Microscopy Sciences,目录号:19150),储存温度:RT
  24. 磷酸氢二钠(Na 2 HPO 4 )(默克,目录号:1.06586.1000),储存温度:室温(RT)(用于配方1-) 3)
  25. 环氧包埋介质,固化剂DDSA:C 16 H 26 O 3 (Sigma-Aldrich,目录号:45346),储存温度:RT(在食谱6中使用)
  26. 环氧包埋介质,固化剂MNA:C 10 H 10 O 3 (Sigma-Aldrich,目录号:45347),储存温度:RT(在食谱6中使用)
  27. 环氧嵌入介质:Epon TM 812替代品(Sigma-Aldrich,目录号:45345),储存温度:RT(用于配方6)
  28. 用于合成的水溶液中的戊二醛(25%):C 5 H 8 O 2 (Merck,目录号:8.20603.1000),储存温度:4°C(用于配方1)
  29. 盐酸(HCl)(Sigma-Aldrich,目录号:H1758),储存温度:RT(用于配方2)
  30. 硝酸铅(II):( PbNO 3 ) 2 (PanReac AppliChem,目录号:131473),储存温度:RT(用于配方8)
  31. N-苄基二甲胺:C 9 H 13 N(Sigma-Aldrich,目录号:185582),储存温度:RT(用于配方6)
  32. 多聚甲醛:(CH 2 O)n(Merck,目录号:1.04005.1000),储存温度:RT(用于配方1)
  33. 苦味酸溶液(1.3%)溶于H 2 O(饱和):( O 2 N) 3 C 6 H 2 OH(Sigma-Aldrich,目录号:P6744),储存温度:RT(用于配方1)
  34. 聚(乙二醇)(PEG):C 2n H 4n H 2 O n + 1 (Sigma- Aldrich,目录号:202444),储存温度:RT
  35. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541),储存温度:RT(用于配方2)
  36. 磷酸二氢钾(KH 2 PO 4 )(Sigma-Aldrich,目录号:229806),储存温度:RT(用于配方2)
  37. 氯化钠(NaCl)(PanReac AppliChem,目录号:131659.1211),储存温度:RT(用于配方2-4)
  38. 氢氧化钠颗粒(PanReac AppliChem,目录号:131687.1211),储存温度:RT(用于配方8)
  39. 磷酸二氢钠一水合物(NaH 2 PO 4 •H 2 O)(PanReac AppliChem,目录号:131965.1211),储存温度:RT(用于食谱1-3)
  40. 柠檬酸三钠2水合物:Na 3 C 6 H 5 O 7 •2H 2 O(默克,目录号:6448),储存温度:4°C(用于配方8)
  41. Triton X-100(Sigma-Aldrich,目录号:X100),储存温度:RT(用于配方5)
  42. Trizma ®碱:NH 2 C(CH 2 OH) 3 (Sigma-Aldrich,目录号:T1503 ),储存温度:RT(用于配方4和5)
  43. Trizma ®盐酸盐:NH 2 C(CH 2 OH) 3 •HCl(Sigma-Aldrich,目录号:T3253),储存温度:RT(用于配方4)
  44. 乙酸双氧铀:UO 2 (CH 3 COO) 2 (电子显微镜科学,目录号:22400),储存温度:RT(用于食谱7)
  45. 固定解决方案(见食谱)
  46. 磷酸盐缓冲盐水(PBS 1x)(见食谱)
  47. 0.1 M磷酸盐缓冲液(0.1 M PB)(见食谱)
  48. 三氯化氢缓冲盐水(TBS 1x)(见食谱)
  49. 含Triton X-100(TBST)的Tris缓冲盐水(见食谱)
  50. Epon树脂(见食谱)
  51. 乙酸双氧铀(2%,水溶液)染色溶液(见食谱)
  52. Reynold的柠檬酸铅染色解决方案(见食谱)

设备

  1. Erlenmeyer烧瓶(Sigma-Aldrich,目录号:Z567868)
  2. Shaker(Heidolph,Duomax 1030)
  3. 烤箱(Grupo Selecta,Dryterm)
  4. Vibratome(Leica Biosystems,型号:Leica VT1000 S)
  5. 超级白金刀(吉列)
  6. Ultramicrotome(RMC产品,型号:PowerTome XL)
  7. Histo 6毫米金刚石刀45°(硅藻土)
  8. 超2.4毫米钻石刀45°(硅藻土)
  9. 透射电子显微镜(飞利浦,型号:EM208S)
  10. 数码Morada相机(奥林巴斯SIS Morada相机)
  11. 冷冻固定装置(Reichert-Jung,型号:KF 80)
  12. 冻结替代系统(Reichert AFS)

软件

  1. Adobe Photoshop(CS3,Adobe Systems,San Jose,CA,USA)
  2. ImageJ(NIH,USA; RRID:SCR_003070)
  3. GraphPad Prism 5(GraphPad Software Inc.,San Diego,USA; RRID:SCR_002798)

程序

  1. 预嵌入(图1)
    保存脑组织
    1. 通过腹膜内注射氯胺酮/甲苯噻嗪(80 / 10mg / kg体重)麻醉动物(至少n = 3)。
    2. 在室温下用PBS(0.1 M,pH 7.4,20-25°C)通过左心室灌注动物~20 s,然后用4%甲醛(从多聚甲醛中新解聚)制成的冰冷固定液,0.2%苦味酸和PB中的0.1%戊二醛(0.1M,pH7.4)。固定液/小鼠:250毫升。灌注时间15分钟。固定液/大鼠:500毫升。灌注时间:30分钟。
    3. 从颅骨中取出大脑,并在4°C下固定在固定液中约1周。将样品储存在含有0.025%叠氮化钠的0.1 M PB稀释的固定剂(1:10)中,在4°C下使用。


    图1.嵌入免疫电子显微镜技术前后的一般步骤的时间表 s

    电子显微镜预嵌入免疫金法 (图1,2A和3A)
    1. 在振动切片机上切下50μm厚的切片,并在室温下置于0.1M PB(pH 7.4)的12孔细胞培养板中。来自每个脑的切片在分开的板中,在4℃下在含有0.025%叠氮化钠的0.1M PB(pH 7.4)中储存。选择包含感兴趣区域的每个脑的两个或三个切片并放置在新的平板中。每孔总体积:1毫升。
    2. 在摇床(300rpm)上在室温下在TBS 1x(pH 7.4)中含有10%BSA,0.02%皂苷和0.1%叠氮化钠的封闭溶液(1ml /孔)中预孵育30分钟。
    3. 在摇床上与在含有0.004%皂苷和0.1%叠氮化钠的10%BSA / TBS 1x中稀释的山羊多克隆抗CB 1 受体抗体(1:100稀释,1ml /孔)孵育。将培养皿放置在轨道振荡器上,在4°C下放置两天。 
    4. 在1%BSA / TBS中洗涤5次(3×1分钟和2×10分钟)。
    5. 在摇动器上与含有0.004%皂苷的1%BSA / TBS中的1.4nm金缀合的兔抗山羊IgG(Fab'片段,1:100,Nanoprobes Inc.,Yaphank,NY,USA; 1ml /孔)孵育在室温下3小时。
    6. 在1%BSA / TBS中洗涤三次(每次10分钟)。将组织在1%BSA / TBS中在振荡器上保持4℃过夜。
    7. 用TBS(1ml /孔)在室温下制备1%戊二醛后固定10分钟。
    8. 用双蒸水洗涤三次(每次10分钟)。 
    9. 将切片转移到玻璃试管中。
    10. 在黑暗中用HQ Silver试剂盒(Nanoprobes Inc.,Yaphank,NY,USA; 1ml /管)强化金颗粒12分钟。 
    11. 用双蒸水洗涤三次(每次1分钟)。 
    12. 在0.1 M PB(pH 7.4)中洗涤三次(每次10分钟)。 
    13. 将切片转移到玻璃瓶(15毫升,3×5厘米)。
    14. 在黑暗中将Osmicate(1%四氧化锇在0.1M PB,pH7.4; 1ml /小瓶中)保持20分钟。
    15. 在0.1M PB(pH 7.4)中洗涤三次(每次10分钟)。
    16. 在分级乙醇中脱水(50%,70%,96%;每次5分钟),然后在100%乙醇(每次5分钟)(1ml /小瓶)中脱水三次。 
    17. 用环氧丙烷(3×5分钟,1毫升/小瓶)代替。
    18. 用振荡器在室温下用环氧丙烷和Epon树脂812(1ml /小瓶)的1:1混合物渗透切片过夜。 
    19. 与纯Epon树脂812(1ml /小瓶)混合,用于>在室温下2小时。
    20. 将部分放在两个载玻片之间,用铝箔包裹载玻片。
    21. 在60°C下聚合树脂嵌入部分2天。
    22. 在超薄切片机上切割1μm半薄切片。
    23. 修剪块并用金刚石刀切割60纳米超薄切片并收集在镍网格上。 
    24. 在室温下用2.5%柠檬酸铅(1滴/网格)染色切片20分钟。 
    25. 用双蒸水(1滴/格)洗涤三次(每次10分钟)。
    26. 在飞利浦EM208S透射电子显微镜下检查。 
    27. 使用数字Morada相机(Olympus)拍摄部分。

    双重预嵌入免疫金和免疫过氧化物酶法 (图1,2B和3B-3D)
    1. 在振动切片机上切下50μm厚的切片,并在室温下在0.1M PB(pH 7.4)中的12孔细胞培养板中收集。来自每个脑的切片在分开的板中,在4℃下在含有0.025%叠氮化钠的0.1M PB(pH 7.4)中储存。选择包含感兴趣区域的每个脑的两个或三个切片并放置在新的平板中。每孔总体积:1毫升。
    2. 在摇床(300rpm)上在TBS 1x,pH 7.4中含有10%BSA,0.02%皂苷和0.1%叠氮化钠的封闭溶液(1ml /孔)中在室温下孵育30分钟。 
    3. 用山羊多克隆抗CB 1 受体抗体或豚鼠多克隆抗CB 1 受体抗体(1:100,1 ml /孔稀释)与任一组合孵育切片小鼠单克隆抗GFAP抗体(1:1,000),兔多克隆抗GLAST抗体(0.3μg/ ml),或小鼠单克隆抗 - gephyrin抗体(1:250),用含有0.004%皂苷的10%BSA / TBS 1x制备和0.1%叠氮化钠。将培养皿置于4°C的轨道振荡器上2天。
      注意:豚鼠多克隆抗CB 1 抗体用于兔多克隆抗GLAST抗体的双重免疫标记。
    4. 在1%BSA / TBS中洗涤5次(3×1分钟和2×10分钟)。
    5. 与相应的生物素化的二抗(1:200)和1.4nm金偶联的第二兔抗山羊IgG(Fab'片段,1:100,Nanoprobes Inc.,Yaphank,NY,USA)或1.4nm金偶联的二级山羊一起孵育将抗豚鼠IgG(Fab'片段,1:100,Nanoprobes Inc.,Yaphank,NY,USA)在摇床上在含有0.004%皂苷的1%BSA / TBS中在室温下稀释4小时。
    6. 在室温下用振荡器在1%BSA / TBS(每次10分钟)中洗涤三次。 
    7. 将在洗涤溶液(1ml /孔)中制备的抗生物素蛋白 - 生物素 - 过氧化物酶复合物(ABC)(1:50)在室温下孵育1.5小时。 
    8. 在1%BSA / TBS中洗涤三次(每次10分钟)。将组织置于1%BSA / TBS中,在振荡器上于4℃保持过夜。
    9. 在TBS(1ml /孔)中用1%戊二醛在室温下固定10分钟。 
    10. 用双蒸水洗涤三次(每次10分钟)。 
    11. 切片转移到试管中。
    12. 在黑暗中用HQ Silver试剂盒(Nanoprobes Inc.,Yaphank,NY,USA; 1ml /管)强化金颗粒12分钟。 
    13. 用双蒸水洗涤三次(每次1分钟)。 
    14. 在0.1 M PB(pH 7.4)中洗涤三次(每次10分钟)。 
    15. 将切片转移到玻璃小瓶(15ml,3×5cm)中。
    16. 在室温下,在0.1M PB(1ml /小瓶)中制备的0.05%DAB和0.01%过氧化氢孵育3分钟。 
    17. 在0.1 M PB(pH 7.4)中洗涤三次(每次10分钟)。 
    18. 在黑暗中将Osmicate样品(1%四氧化锇在0.1M PB,pH7.4; 1ml /小瓶中)保持20分钟。
    19. 在0.1M PB(pH 7.4)中洗涤三次(每次10分钟)。
    20. 在分级乙醇中脱水(50%,70%,96%;每次5分钟),然后在100%乙醇(每次5分钟)(1ml /小瓶)中脱水三次。 
    21. 用环氧丙烷(3×5分钟,1毫升/小瓶)代替。
    22. 用振荡器在室温下用环氧丙烷和Epon树脂812(1ml /小瓶)的1:1混合物渗透切片过夜。 
    23. 与纯Epon树脂812(1ml /小瓶)混合,用于>在室温下2小时。
    24. 将部分放在两个载玻片之间,并将载玻片包裹在铝箔中。
    25. 将树脂嵌入部分在60°C的烘箱中聚合2天。 
    26. 在超薄切片机上切割1μm半薄切片。
    27. 修剪块并用金刚石刀切割60纳米超薄切片并收集在镍网格上。 
    28. 在室温下用2.5%柠檬酸铅(1滴/网格)染色切片20分钟。 
    29. 用双蒸水(1滴/格)洗涤三次(每次10分钟)。
    30. 在飞利浦EM208S透射电子显微镜下检查。 
    31. 使用数字Morada相机(Olympus)拍摄部分。

  2. 后嵌入式(图1)
    使用嵌入后免疫金法进行单/双标记 (图1,2C和3E-3F)
    1. 保存脑组织。为获得最佳超微结构,如上所述麻醉并灌注动物。 
    2. 从颅骨中取出大脑,并在4°C下固定在固定液中约1周。然后,将大脑置于0.1 M PB稀释的固定剂(1:10)加0.025%叠氮化钠中,温度为4°C,然后用PB冲洗直至下一步。 
    3. 样品修剪(从感兴趣的区域切割0.5 x 0.5 x 1 mm的小矩形片)。
    4. 使用冷冻固定装置(KF80; Reichert,Vienna,Austria)在甘油中冷冻保护(PB中10%,20%和30%)并在液体丙烷中快速冷冻。 
    5. 用甲醇和0.5%乙酸双氧铀冷冻替代。
    6. 嵌入Lowicryl HM20(Lowi,Waldkraiburg,Germany)。

    免疫金标记程序
    1. 在超薄切片机上切割1μm半薄切片。
    2. 在镍单槽formvar涂层网格上收集70纳米超薄切片。
    3. 用含有0.1%NaBH 4 和50 mM甘氨酸(10μl滴/网格)的TBST洗涤样本10分钟。 
    4. 在TBST中冲洗3次(10μl滴/网格)(每次1分钟)。
    5. 在含有10%BSA的TBST的封闭溶液(10μl滴/网格)中预孵育10分钟。
    6. 用抗CB 1 受体(1:50)或抗mGluR2 / 3(1:50)抗体或用2%BSA制备的它们的混合物孵育(10μl滴/网格)/ TBST,在4°C过夜。 
    7. 在TBST中洗涤三次(10μl滴/网格)(每次10分钟)。
    8. 在含有10%BSA和0.5%PEG的TBST中的封闭溶液(10μl滴/网格)中预孵育10分钟。 
    9. 用不同金粒径的二抗孵育(10μl滴/网格):驴18nm胶体金偶联亲和纯化的抗山羊IgG(用于CB 1 )和山羊F(ab')<将sub> 2 抗兔IgG与10nm胶体金颗粒(对于mGluR2 / 3)偶联,在室温下在2%BSA / TBST中1:20稀释2小时。
    10. 在双蒸水中洗涤三次(10μl滴/格)(每次10分钟)。
    11. 用2%乙酸双氧铀(20分钟)和2.5%柠檬酸铅(2分钟)(10μl滴/格)对抗。
    12. 用双蒸水(10μl滴/格)洗涤三次(每次10分钟)。
    13. 在飞利浦EM208S透射电子显微镜下检查。&nbsp;
    14. 使用数码Morada相机(奥林巴斯)拍摄标本。


    图2.说明高分辨率电子显微镜的三种免疫标记方法的示意图


    图3. CB 1 受体免疫定位在啮齿动物脑的不同亚细胞区室。单一预嵌入免疫金(A)和双预嵌入免疫金和免疫过氧化物酶方法(BD) 。 A.在与树突(den,紫色)相邻的突触前GABA能末端(ter,黄色)处的CB 1 受体标记(箭头)。 CB 1 受体颗粒定位于与脊柱(sp,紫色)相关的突触前谷氨酸能末端(ter,粉红色)。线粒体(m,红色)在谷氨酸能(ter,粉红色)和GABA能(ter,黄色)突触前末梢(CA1 stratum radiatum,成年小鼠海马)中表现出CB 1 受体免疫标记。 B. CB 1 受体标记(箭头)在突触前GABA能末端(ter,黄色),谷氨酸能末端(ter,紫色)和一个星形胶质细胞分支(白色箭头; as,绿色)中的小鼠梨状皮质。用抗GLAST /免疫过氧化物酶/ DAB方法(as中的黑色沉淀物)标记星形胶质细胞。 C. CB 1 受体标记(箭头)在突触前GABAergic末端(ter,黄色)与树突(den,紫色)相邻并在一个星形胶质细胞过程中(白色箭头;为绿色)小鼠齿状回的分子层。用抗GFAP /免疫过氧化物酶/ DAB方法(as中的黑色沉淀物)标记星形胶质细胞。 D.突触前末端(ter,黄色)的CB 1 受体标记(箭头)与抗gephyrin /免疫过氧化物酶/ DAB方法(黑色沉淀在den,紫色)结合,以正向鉴定抑制性突触后膜GABA能神经突触白色箭头:CB 1 受体标记在填充细胞间隙(大鼠前肢皮质)的薄星形细胞过程中。 E-F。双嵌入后免疫金法揭示突触前CB 1 受体(ter; 18 nm直径金颗粒)和突触后mGluR2 / 3(den,sp; 10 nm直径金颗粒)的抑制作用(ter,yellow)和小鼠齿状分子层中的兴奋性(ter,pink)突触。比例尺= 500 nm。

数据分析

  1. 预嵌入法中CB 1 受体免疫标记的半定量分析
    为了使标准条件最大化,将预嵌入免疫金方法同时应用于从研究中的动物获得的所有切片(至少n = 3)。对每只动物进行三次重复实验。
    &NBSP;首先在光学显微镜下观察免疫金标记的树脂包埋的振动切片部分,以选择可重复的部分感兴趣区域(即,CA1海马,齿状分子层,前肢皮质或梨状皮质)。 CB 1 受体免疫标记。然后,切割来自树脂包埋组织的半薄切片,并将前五个超薄切片收集到两个网格上。为了进一步标准化不同动物之间的条件,仅收集每个样本表面的前1.5μm并随机拍照。对于所有研究的动物,总是小心地并且以相同的方式进行取样。为避免偏见,研究人员在拍摄和分析电子显微照片时仍然失明。
    &NBSP;兴奋性和抑制性突触通过其超微结构特征鉴定;兴奋性突触与突触后密度和突触前轴突末端不对称,包含丰富,清晰和球形的突触小泡。抑制性突触与细长的突触后膜和含有多形性突触小泡的轴突末端对称。由于缺乏突触后膜密度,除非连续切片完成,否则突触的抑制性质可能会产生误导。避免这种情况的替代方案是使用抗gephyrin的抗体,gephyrin是抑制性突触的突触后锚定蛋白标记,其可用于明确鉴定抑制性突触。在含有GFAP或GLAST DAB免疫沉积物的星形胶质细胞过程中评估星形胶质细胞中的CB 1 受体。
    &NBSP;然后将如上所述鉴定的不同区室上的CB 1 受体标记的比例制成表格。当至少一个CB 1 受体免疫颗粒在所研究的特定区室的膜的~30nm内时,并且在线粒体标记的情况下来自其他膜的≥80nm时,考虑阳性标记。然后计数金属颗粒并通过ImageJ软件通过测量它们的膜长度来确定阳性隔室中的CB 1 受体密度(颗粒/μm膜)。我们还估计了CB 1 受体免疫颗粒在不同谱中的比例与总CB 1 受体表达的比值。这提供了关于特定脑区域的不同区室(兴奋性和抑制性突触,星形胶质细胞,线粒体,其他细胞区室)中CB 1 受体分布的信息。对于星形胶质细胞,还计算从星形细胞CB 1 受体免疫颗粒到最近的突触的距离,以确定受体如何在三联突触的背景下分布。为此,识别CB 1 受体阳性星形胶质细胞周围的附近突触,测量距离(ImageJ软件),选择的星形胶质细胞免疫颗粒的最近突触,以及来自所有最近突触的数据和分析。
    &NBSP;所有值均以平均值±S.E.M表示。使用统计软件包(GraphPad Prism 5,GraphPad Software Inc.,San Diego,USA)。在运行统计检验之前,始终应用常态检验(Kolmogorov-Smirnov正态性检验)。使用参数或非参数双尾学生 t - 测试或单向ANOVA以及随后的事后分析(Bonferroni post-test)分析数据。

  2. 在嵌入后方法中定量CB 1 受体免疫标记
    鉴定的兴奋性和抑制性突触末端的电子显微照片是从特定脑区域(例如,齿状回的分子层)随机获得的。为了分析,从所研究的每只动物中选择包含完整质膜,突触小泡和突出突触裂缝标准的50个突触。为避免偏见,研究人员在拍摄和分析电子显微照片时仍然失明。使用统计软件包分析CB 1 受体阳性兴奋性和/或抑制性末端的百分比以及接受CB 1 阳性突触前末梢的mGluR2 / 3标记的突触后树突的百分比。 (GraphPad Prism 5,GraphPad Software Inc.,San Diego,CA,USA)。如预嵌入技术(例如,膜接近)中所述,标记被认为是阳性的。数据显示为平均值±S.E.M。如前所述计算正极端子中的CB 1 受体密度(颗粒/μm膜)。另外,为了确定突触后元件中mGluR2 / 3(或任何其他受体)相对于突触前释放位点的精确亚突触分布,测量金颗粒频率的分布。此外,可以确定突触前boutons中的亚突触CB 1 受体分布。在这种情况下,金颗粒的突触或周/突外定位是根据其从突触后密度和突触前活动区边缘获得的60 nm宽片段中的分配来定义的(边缘定位= 0) 。如预嵌入方法部分所述进行统计分析。

食谱

  1. 固定解决方案
    在微波炉中加热600毫升蒸馏水至60°C
    加入11.5克Na 2 HPO 4 (无水)(MW:141.96)
    加入40g多聚甲醛(MW:30.03)并在60℃下摇动。可能需要30分钟才能解散
    加入2.62 g NaH 2 PO 4 •H 2 O(MW:137.99)
    将溶液过滤到锥形瓶中,加入2ml饱和苦味酸(MW:229.11) 用蒸馏水补足1升
    冷却并储存在4°C直至使用
    在灌注前,加入4毫升戊二醛(25%)
  2. 磷酸盐缓冲盐水(PBS 1x)(0.1M,pH7.4)
    对于1L PBS 1x,准备如下:
    从800毫升蒸馏水开始:
    加入8克NaCl(分子量:58.44)
    加入0.2克KCl(分子量:74.55)
    加入1.44克Na 2 HPO 4 (MW:141.96)
    加入0.24克KH 2 PO 4 (MW:136.09)
    用HCl(MW:36.46)将pH调节至7.4
    加入蒸馏水至总体积为1升
  3. 0.1M磷酸盐缓冲液(0.1M PB)pH = 7.4
    储备溶液:0.2M PB,pH = 7.4
    对于1升0.2 M PB,准备如下:
    加入5.24克NaH 2 PO 4 •H 2 O(MW:138)
    加入23.0克Na 2 HPO 4 (MW:141.96)
    蒸馏水,补充1升溶液 为了制备0.1M PB,将原液0.2M PB在蒸馏水中1:1稀释
  4. 三氯化氢缓冲盐水:3M NaCl + 1M Tris-HCl,pH = 7.4; (TBS 1x)
    储备溶液:0.3M NaCl + 0.1M Tris-HCl,pH = 7.4(10x TBS)
    对于1升10x TBS,准备如下:
    加入175克NaCl(分子量:58.44)
    加入19.4克Trizma碱(分子量:121.14)
    加入132.2克Trizma-HCl(MW:157.60)
    用蒸馏水补充1升溶液
    为制备1L TBS 1x,将TBS 10x储备溶液在蒸馏水中1:9稀释
  5. 含Triton X-100(TBST)的Tris缓冲盐水
    取450毫升蒸馏水
    加入3.03克Trizma碱(分子量:121.14)
    加入4.5克NaCl(分子量:58.44)
    将pH调节至7.4,用蒸馏水将溶液调至500 ml
    加入0.5毫升Triton X-100(分子量:646.86)
  6. Epon树脂
    加入81.3克EPON 812(分子量:178.18)
    加入53.0克EPONHÄRTERDDSA(MW:266.38)
    加入35.7克MNA(分子量:178.18)
    加入2.24ml N-苄基二甲胺Fluka 13370(MW:135.21)
    混合至少2小时,然后在-20℃下储存在注射器中
  7. 醋酸铀酰(2%,水溶液)染色溶液
    在2ml蒸馏水中制备0.04g乙酸双氧铀(UO 2 (CH 3 COO) 2 (MW:388.11)),离心机于11,600 xg 5分钟并使用上清液
  8. Reynold的柠檬酸铅染色解决方案
    加入2.66g硝酸铅(Pb(NO 3 ) 2 ,MW 331.2)和3.52g柠檬酸三钠二水合物(Na 3 C 6 H 5 O 7 •2H 2 O,MW:294.10)在84毫升双蒸水中(当加入柠檬酸钠时,溶液变得混浊是正常的)
    在20ml双蒸水中制备0.8g 1N NaOH(MW:39.99)
    向柠檬酸铅溶液中加入16毫升NaOH溶液(当加入NaOH时溶液变澄清)
    过滤溶液以去除任何未溶解的物质

致谢

这项工作得到了巴斯克政府的支持(BCG IT764-13); MINECO / FEDER,UE(SAF2015-65034-R); Red de Trastornos Adictivos,Instituto de Salud Carlos III(ISC-III)和欧洲区域发展基金 - 欧盟(ERDF-EU)(RD16 / 0017/0012)。我们要感谢Niels Christian Danbolt教授为我们提供兔多克隆抗A522 EAAT1 [GLAST]抗体和Mahmood Amiry-Moghaddam教授和BjørgRiber教授的Lowicryl HM20嵌入(分子医学系解剖学部,挪威奥斯陆大学基础医学研究所。

利益争夺

作者声明没有利益冲突或竞争利益。

伦理

动物护理和使用议定书得到了巴斯克大学动物福利伦理委员会(CEEA / M20 / 2016/073; CEIAB / 2016/074)的批准,并符合欧洲共同体理事会的指令。 2010年9月22日(2010/63 / EU)和西班牙法规(Real Decreto 53/2013,BOE 08-02-2013)。

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引用:Puente, N., Bonilla-Del Río, I., Achicallende, S., Nahirney, P. C. and Grandes, P. (2019). High-resolution Immunoelectron Microscopy Techniques for Revealing Distinct Subcellular Type 1 Cannabinoid Receptor Domains in Brain. Bio-protocol 9(2): e3145. DOI: 10.21769/BioProtoc.3145.
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