The technique allows the confirmatory diagnosis of prion diseases by the technique PrP scrapie in tissues, analyzing the PrP scrapie types and distribution to evaluate punitive deviation to the known PrP scrapie deposition profile. The technique is standardized and uses specific antibodies to PrP scrapie, enabling the visualization of the PrP scrapie deposition in tissues. The technique also gives information about the neuroanatomical area and type of cells.
Begin by placing the slides with the tissue sections in a stainless-steel staining basket. Emerge the basket in a xylene bath. After three minutes, remove and immerse the basket once again.
To remove xylene and rehydrate the sections, immerse the basket in an absolute ethanol bath. After three minutes, remove and immerse the basket once again. Air dry the sections before placing them in a 90%ethanol bath for one minute.
Next, transfer the section to a 70%ethanol bath for another minute. For each ethanol bath treatment, gently agitate the slide basket twice and drain the basket prior to the next transfer. Transfer the basket to a 50%ethanol bath for one minute.
Carefully immerse the tissue sections in 98%formic acid at room temperature for 30 minutes. Rinse the sections in tap water for five 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 2 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. Bridge the sections under running water for five minutes After draining, immerse the sections in Tris-buffered saline for an additional five minutes. For immunodetection, place each slide onto a commercially available cover-plate holder pre-moistened 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 pre-incubating 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 five 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. The level of non-specific immunolabeling in known transmissible spongiform encephalopathies-negative control animals was determined to interpret the specific abnormal prion proteins immunolabeling properly. Non-target labeling by anti-PrP antibodies was noted to occur as discrete intraneuronal fine-particulate staining, irregular fine linear threads of stain in the neuropil, diffuse non-particulate labeling in some gray and white matter areas, and diffuse cytoplasmic labeling of neurons.
The table summarizes the description of immunolabeled abnormal prion protein types observed in bovine spongiform encephalopathy, classical and atypical scrapie, and a chronic wasting disease of cervids for a positive diagnosis and established criteria for negative, inconclusive, and unsuitable results. All the steps are very important. The pH of the solutions and the room temperature are important features for the success of this technique.
Developing immunohistochemistry to detect both PrP scrapie and type of cells can give information about each cell involved in prime pathogenesis.
