Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry
Trascrizione
Take a slide with a fixed mammalian brain section exhibiting misfolded prion protein aggregates.
Treat the section with formic acid to reduce the infectivity of the prion aggregates.
Treat with an acidic buffer inside a hydrated pressure chamber. A high heat and acidic pH break fixation-induced protein crosslinks, unmasking the antigenic epitopes.
Immerse in hydrogen peroxide to inactivate endogenous peroxidase, preventing undesired background staining.
Place the slide in a cover plate and add a buffer to maintain tissue hydration.
Introduce a blocking solution, preventing non-specific antibody binding.
Add a primary antibody specific to the prion aggregates.
Remove the unbound antibody molecules. Introduce a biotinylated secondary antibody that binds to the primary antibody.
Remove the unbound antibody molecules. Add a biotinylated peroxidase enzyme complexed with avidin that binds to biotin on the secondary antibody.
Add a chromogenic substrate, which the peroxidase oxidizes into a colored product.
Using a microscope, observe the stained aggregates.
