Multiplex Immunohistochemistry for Uterine NK Cell Subtype Analysis in Endometrial Tissues

To begin, obtain the human endometrial tissue section on a glass slide for multiplex immunohistochemistry, and bake the tissue sections at 60 degrees Celsius for two hours. Subsequently, immerse the slide sequentially in appropriate solutions for dewaxing, hydration, and fixation. Add 200 milliliters of the respective AR6 or AR9 buffer for each antibody to a heat-resistant antigen retrieval cassette.

After placing the sections in the antigen retrieval buffer, heat them in a microwave on high power until boiling. Cover the cassette loosely, and heat on low power for 15 minutes. After the slide cools down, rinse it with double distilled water, followed by PBST.

Then remove the excess solution with absorbent paper, and draw a complete circle around the tissue with a hydrophobic barrier pen. Then incubate the sections with 3%hydrogen peroxide for eight minutes at room temperature. Next, apply a blocking solution, and incubate for 10 minutes at room temperature.

After removing the blocking solution, apply 150 microliters of the first primary antibody, CD49a, to each slide at a dilution of 1 to 1000 in 1x antibody diluent. Before applying the secondary antibody, remove excess moisture using absorbent paper. Add four to five drops of the 1x anti-mouse and rabbit horseradish peroxidase secondary antibody onto each slide.

Then dilute the tyramide signal amplification or the TSA dye 1 to 100 times in the amplification diluent. And add 150 microliters of this diluted solution to each slide and incubate. To eliminate unbound fluorophores, boil the sections in 200 milliliters of AR6 buffer within a heat resistant cassette in a microwave for two minutes.

Then turn the microwave from high power to low power for 15 minutes. Add 150 microliters of DAPI working solution diluted in PBST dropwise. Dry the sections entirely in a dark area for approximately 30 minutes.

Apply a single drop of the anti-quenching fluorescent mountant, and carefully place a coverslip.