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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.
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