Performing Immunohistochemistry on a Non-Human Primate Brain Tissue

Begin with a chemically fixed non-human primate brain section.

The aldehyde-based fixative cross-links proteins, preserving tissue structure, but leaves some reactive aldehyde residues.

Wash to remove the cryoprotectant layer surrounding the tissue section.

Add a reducing agent to neutralize the reactive aldehyde groups, producing reaction by-products.

Wash to remove these by-products and excess reducing agents.

Apply a blocking solution to prevent non-specific antibody binding.

Add primary antibodies targeting a protein expressed on specific sites on the neuronal membrane. 

Wash to remove unbound antibodies.

Add biotinylated secondary antibodies targeting the primary antibodies. 

Wash to remove unbound antibodies.

Overlay with avidin-biotin-peroxidase complexes that bind to the secondary antibodies.   

Wash again to remove unbound complexes.

Introduce a chromogenic substrate along with hydrogen peroxide. In the presence of hydrogen peroxide, the peroxidase enzyme oxidizes the substrate, forming brown precipitates.

Begin by transferring 50 micron-thick sections of acrolein fixed non-human primate brain, containing the region of interest to a 12-well plate containing PBS. Wash the free floating sections three times in PBS for five minutes at room temperature. Then incubate the sections in freshly prepared sodium borohydride solution for 30 minutes at room temperature. Rock gently without a cover.

Once the 30 minutes have elapsed, aspirate the sodium borohydride and add PBS to each well. Place the plate on a rocker and shake vigorously for 10 minutes. Repeat the wash two more times, or until none of the reaction gas remains. Block the sections by incubating in a solution of 2% normal serum and 0.5% cold fish gelatin, diluted in PBS for one hour at room temperature with gentle rocking.

Once the incubation time has elapsed, incubate the sections and primary antibody solution with gentle rocking overnight at room temperature. The next day, after washing the sections as before, incubate the sections in secondary antibody solution for 1.5 hours at room temperature with gentle rocking. After washing the sections as before, incubate in avidin biotin peroxidase, or ABC solution, for one hour with rocking.

Next, prepare a fresh solution of 0.05% 3,3-diaminobenzidine with 0.005% hydrogen peroxide diluted in TBS. After washing, incubate sections in the DAB solution for three to seven minutes with gentle rocking. Once the brown precipitate has developed to the desired color, stop the reaction by quickly washing twice in cold TBS, then through a series of timed TBS and PB washes.