Detecting Abnormal Prion Proteins in Brain Tissue Using Immunohistochemistry

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.

Carefully immerse the tissue sections in 98% formic acid at room temperature for 30 minutes. Rinse the sections in tap water for 5 minutes, followed by rinsing twice in distilled water. Use a stainless steel staining container in the pressure chamber filled with 500 milliliters of distilled water to pretreat 10 millimolar citrate buffer at 98 degrees Celsius for 20 minutes.

For quality control purposes, place a section of adhesive autoclave indicator tape on the basket to monitor the temperature and pressure. Once the alarm indicates that the equipment has attained the program time and temperature, immerse the basket with slides. Next, initiate the program on the pressure chamber to set point two, and record the initial and final pressure of this program.

For the inactivation of endogenous peroxidase, immerse the slide basket containing the sample sections in a bath of 3% hydrogen peroxide in methanol for 30 minutes. Rinse the sections under running water for 5 minutes. After draining, immerse the sections in tris-buffered saline for an additional 5 minutes. For immunodetection, place each slide onto a commercially available cover plate holder premoistened with Tris-buffered saline.

With the tissue side facing the holder, and slide edges coinciding with the two lower points of the holder, ensure to avoid air bubbles. Hold the slide holder assembly between the thumb and forefinger. Keep one finger on top of the sample slide and the other on the bottom of the holder. Then place the assembly in the gallery of the system.

To ensure that the set is well assembled, fill the well between the sample slide and the holder with tris-buffered saline, without overflowing it. From this point, ensure that approximately 80 microliters of Tris-buffered saline must be retained between the holder and the slide. Do not allow the sections to dry.

Decrease the background staining prior to the treatment with the primary antibody by preincubating the slide samples for 30 minutes with 20% normal serum from the same species as the secondary antibody host in Tris-buffered saline. Without washing the sections, apply 200 microliters of the primary antibody solution directly into each well of the slide holder set, and incubate for 60 minutes at room temperature.

To wash the sections, fill the wells with tris-buffered saline, and wait for 5 minutes. Repeat the wash twice. Next, dilute the biotinylated secondary antibody at 1 to 200 concentration in tris-buffered saline with 10% horse serum.

Repair the required volume, depending on the number of sections to be treated. Apply 200 microliters of secondary antibody solution to each well of the slide holder set. After 30 minutes of incubation at room temperature, repeat the Tris-buffered saline wash.

For incubation with avidin-biotin complex peroxidase, prepare the reagent 30 minutes before use. and apply 200 microliters of the solution in each well of the slide plate holder. Once done, incubate the plate holder set for 30 minutes at room temperature.