This article describes a protocol for immunohistochemical staining of human rectal biopsy sections to detect S-100 protein, a marker associated with Hirschsprung's disease. The method involves several steps including blocking, antibody incubation, and visualization techniques to highlight the presence of the target protein.
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 washes in PBS and add 50 microliters of polymer enhancer to each slide. After 20 minutes at 37 degrees Celsius, wash the slides three times in PBS, and add 50 microliters of secondary antibody B to each tissue section.
After 30 minutes at 37 degrees Celsius, rinse the sections with three five-minute washes in PBS, and add a drop of freshly prepared DAB working solution to each slide. When the appropriate level of brown staining is detected by light microscopy, rinse the slides in PBS for five minutes, and counterstain the nuclei with one drop of hematoxylin for one minute.
Remove the excess stain under a trickle of water for approximately 30 to 40 seconds, followed by a 25 to 30-second wash in fresh PBS. Differentiate the tissues in 1% hydrochloric acid ethanol for 10 seconds, followed by a one-minute PBS wash.
Dehydrate the slides with five-minute immersions in the reverse order of hydration, and expose the slides to two five-minute changes of xylene. Then, mount the coverslip using ultraclean mounting, and allow the slides to dry before their visualization on a light microscope.
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