Immunohistochemical Staining of S-100 Protein in Human Rectal Suction Biopsy Sections

Take fixed human rectal suction biopsy sections with exposed protein antigens and inactivated endogenous peroxidases.

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

Replace the blocking solution with primary antibodies that bind to the target glial cell protein, S-100, a marker of Hirschsprung's disease.

Remove unbound antibodies and incubate with peroxidase polymers to amplify the signal.

Wash to remove excess polymer. Add secondary antibodies that bind to enzyme polymers and primary antibodies.

Remove unbound antibodies and add a chromogenic substrate.

The peroxidases react with the substrate, forming a colored precipitate.

Rinse to remove excess precipitate.

Add a dye to stain the nuclei.

Wash with an acid-ethanol solution to remove excess dye.

Next, dehydrate the sections using increasing concentrations of ethanol followed by xylene.

Finally, mount and observe under a microscope to visualize the staining indicating the presence of the target protein.

Discard the excess bovine serum albumin, and label the cells with 50 microliters of the primary antibody of interest at 4 degrees Celsius overnight. The next morning, rinse the slides with three three-minute wash