Free shipping starts now, no minimum, no coupons required!

Immunohistochemistry for Brightfield 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 brightfield 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 HRP (horseradish peroxidase).

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 extravidin-peroxidase (Sigma Catalog number E2886) 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 HRP:

1. Incubate sections with horseradish peroxidase labeled donkey anti-rabbit (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. Transfer to 4°C overnight.
2. Rinse sections with 0.02 M PBS containing 2% NDS, 2 x 5 minutes.

 

Detection

1. Incubate sections with a solution of 0.0125% diaminobenzidine (DAB), (Sigma catalog number D5637) containing 0.05% nickel ammonium sulfate for 10 minutes at room temperature.
2. 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.
3. Rinse sections with 0.02 M PBS, 4 x 10 min.
4. Mount sections on glass slides (gelatinized or coated with any other type of adhesive material) and allow to dry.
5. Dehydrate sections in increasing ethanol concentrations (70%, 90%, 100%, 5 minutes in each), delipidate in xylene (10 minutes) and coverslip in Permount (or any other xylene diluted adhesive).

 

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.