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Immunohistochemistry for Fluorescent Microscopy

Immunohistochemistry (IHC) is based on the immunodetection of target proteins in tissue sections. We describe our IHC protocol using rabbit-raised polyclonal primary antibodies on rat floating tissue sections. Immunodetection is accomplished using fluorescent microscopy.

Sacrifice and tissue processing

1. Anesthetize rats with pentobarbital sodium (Pental).
2. Perform transcardial perfusion, first with 50 ml of phosphate buffered saline (0.02 M PBS, pH 7.4), then with 220 ml of ice-cold 4% paraformaldehyde in 0.1 M PBS, pH 7.5 containing 4% sucrose.
3. Cut tissue in coronal blocks and further fix by immersing in the same fixative. Refrigerate overnight.
4. Transfer tissue blocks to 15% sucrose in 0.1 M PBS.
5. Cut tissue in a cryostat within 21 days.
6. Float tissue sections, 30 µm thick in a cryopreservation buffer (40% ethylene glycol and 1% polyvinylpyrrolidone in 0.1 M potassium acetate buffer, pH 6.5), and preserve at -20°C.

 

Staining procedure

1. Rinse floating sections with 0.02 M PBS, 2 x 5 minutes.
2. For antigen retrieval and to quench endogenous peroxidase activity, incubate with 0.2 % hydrogen peroxide in 0.1 M phosphate buffer pH 7.3 containing 0.2% Triton X-100* and 20% methanol for 25 min at room temperature.
3. Rinse sections with 0.02 M PBS, 2 x 5 minutes.
4. Incubate sections with the rabbit primary antibody in a medium containing 0.3% Triton X-100*, 0.05% Tween-20, 2% normal donkey serum (NDS), for 1 hr at room temperature. Transfer to 4°C overnight.
5. Rinse sections with 0.02 M PBS, containing 2% NDS, 2 x 5 minutes.
* If using an “extracellular” antibody decrease Triton X-100 to 0.05%.

Two possible options for secondary antibodies are available here:
A- Secondary antibody conjugated to biotin.
B- Secondary antibody conjugated to a fluorophore.

Option A – Antibody conjugated to biotin:

1. Incubate sections with biotinylated donkey anti-rabbit (Chemicon USA, catalog number AP182B) diluted 1:400 (in 0.02 M PBS, containing 0.3% Triton X-100*, 0.05% Tween-20, and 2% NDS, for 1 hr at room temperature. Transfer to 4°C overnight.
2. Rinse sections with 0.02 M PBS containing 2% NDS, 2 x 5 minutes.
3. Incubate sections with streptavidin-Cy3 (Sigma Catalog number S6402) diluted 1:200 in 0.02 M PBS, for 1 hr at room temperature.
* If using an “extracellular” antibody decrease Triton X-100 to 0.05%.

Option B – Antibody conjugated to a fluorophore:

1. Incubate sections with goat anti-rabbit antibody conjugated to a fluorescent dye diluted 1:200 in 0.02 M PBS, containing 0.3% Triton X-100*, 0.05% Tween-20, and 2% NDS for 1 hr at room temperature. Transfer to 4°C overnight.
2. Rinse sections with 0.02 M PBS containing 2% NDS, 2 x 5 minutes.
* If using an “extracellular” antibody decrease Triton X-100 to 0.05%.

 

Detection

1. Mount sections on glass slides.
2. Allow sections to dry in a fume hood for 1 hr.
3. Stain section on slides with DAPI (Molecular Probes), by placing DAPI solution on each slide, and covering the slide with a piece of parafilm to spread the solution evenly on the slide.
4. After 2 minutes, remove the parafilm and rinse the slide with 0.5 ml distilled deionized water using a pipette (repeat twice).
5. Let slides dry in a fume hood for 30 minutes.
6. Coverslip using the glue ImmuMount (Shandon, UK).
7. Leave slides to dry overnight in the dark.
8. Store at -18º C until confocal microscopy.

 

Antigen Retrieval

If the final result is negative, e.g. no staining, then additional antigen retrieval is recommended. Keep a stock of 0.1% trypsin (Sigma type II, catalog no. T-8128) dissolved in 0.02 M PBS in frozen aliquots. For antigen retrieval, dilute the stock 1:100 to obtain a final trypsin concentration of 0.001%. Add CaCl2 to a final concentration of 0.001%. Incubate the sections in this solution for 5-7 minutes at 37°C and then rinse twice in 0.02 M PBS. Add trypsin-inhibitor (Sigma type II-S, catalog no. T-9128) to the primary antibody solution. Keep a stock of 0.1% trypsin inhibitor diluted in 0.02 M PBS in frozen aliquots. Dilute the stock 1:100 into the primary antibody solution.