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.