Immunohistochemistry

Sacrifice and tissue processing:

Anesthetize rats with pentobarbital sodium (Pental). Fix tissue/organ by transcardial perfusion, first with 50 ml of phosphate buffered saline (0.02 M PBS, pH 7.4) containing heparin (5 U/ml), then with 220 ml of ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.5 containing 4% sucrose. Cut tissue in coronal blocks and further fix by immersing in the same fixative. Refrigerate for 1-2 hrs. Transfer tissue blocks to 12% sucrose in 0.1 M PBS. Section tissue in a cryostat within 10 days. Float tissue sections, 30 µm thick, in 0.1 M PBS and then preserve in a cryopreservation buffer (40% ethylene glycol and 1% polyvinylpyrrolidone in 0.1M potassium acetate buffer, pH 6.5) at -20°C.

 

Staining procedure:

1. Rinse floating sections in 0.02 M PBS, 2x 5 min.
2. Quench endogenous peroxidase activity incubating with 0.2 % hydrogen peroxide in 0.1 M phosphate buffer pH 7.3 containing 0.2% Triton X-100 for 25 min at room temperature.
3. Rinse sections in 0.02 M PBS, 2x 5 min.
4. Incubate sections with the primary antiserum in a medium containing 0.3% Triton X-100, 0.05% Tween 20, 4% normal donkey serum (NDS), for 1 hr at room temperature and then refrigerate overnight.
5. Rinse sections in 0.02 M PBS, containing 4% NDS, 2x 5 min.
6. At this point, staining may proceed with various types of secondary antibodies.
Two alternative procedures are described here:

     Protocol A:

  • Incubate sections with biotinylated donkey anti-rabbit (from Chemicon USA, catalog number AP182B) diluted 1:400 in 0.02 M PBS, containing 0.3% Triton X-100, 0.05% Tween 20, and 4% NDS, for 1 hr at room temperature and then refrigerate overnight.
  • Sections are rinsed in 0.02 M PBS containing 4% NDS, 2x 5 min.
  • For brightfield microscopy, incubate sections with extravidin-peroxidase (Sigma Catalog number E2886) diluted 1:200 in 0.02 M PBS, for 1 hr at room temperature.
  • For fluorescent microscopy, incubate sections with streptavidin-Cy3 (Sigma catalog number S6402) diluted 1:200 in 0.02 M PBS, for 1 hr at room temperature.

     Protocol B:

  • For brightfield microscopy, incubate sections with horseradish peroxidase labeled donkey anti-rabbit (from Chemicon USA, catalog number AP182P), 1:400 in 0.02 M PBS, containing 0.3% Triton X-100, 0.05% Tween 20, and 4% NDS for 1 hr at room temperature and then refrigerate overnight.
  • For fluorescent microscopy, incubate sections with Cy2 labeled goat anti-rabbit (from Jackson ImmunoResearch laboratories, catalog number 111-225-144), diluted 1:200 in 0.02 M PBS, containing 0.3% Triton X-100, 0.05% Tween 20, and 4% NGS for 1 hr at room temperature and then refrigerate overnight.
  • Sections are rinsed in 0.02 M PBS containing 4% NDS, 2x 5 min.
  • For brightfield microscopy: Color development
    • Incubate sections with a solution of diaminobenzidine (DAB), (Sigma catalog number D5637) at the concentration of 0.0125% and containing 0.05% nickel ammonium sulfate for 10 min. at room temperature.
    • Transfer sections to the same DAB solution but with added hydrogen peroxide at a final concentration of 0.0015%. Duration of incubation should be adjusted by the end user.
    • Rinse sections in 0.02 M PBS, 4x 10 min.
    • Mount sections on glass slides (gelatinized or coated by any other type of adhesive material) and allow to dry.
    • Dehydrate sections in increasing ethanol concentrations (70%, 90%, 100%, 5 min. in each), delipidate in xylene (10 min.) and coverslip in Permount (or any other xylene diluted adhesive).
  • For fluorescent microscopy: Mounting, counter-staining, and cover-slipping
        • Mount sections on glass slides from 0.02 M PBS, pH 7.0-7.4.
        • Allow sections to dry in a fume hood for 1 hr.
      • Stain sections with DAPI (Molecular Probes), by placing 240 μl of solution on each slide, and covering the slide with a piece of parafilm to spread the solution evenly on the slide.
      • After 2 min., remove the parafilm and rinse the slide briefly by spraying on it 0.5 ml distilled deionized water from a pippetor (repeat twice).
      • Let slides dry in a fume hood for 30 min.
    • Coverslip using the glue ImmuMount (Shandon, UK).