Encyclopedia of Experiments: Immunology
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Transcript
First, bring the patient serum samples, the biochip slide, the PBS powder, and the Tween-20 from the refrigerated cell-based assay kit to the room temperature. Then, prepare more than 200 milliliters of PBS wash buffer containing 0.2% Tween-20 for each biochip slide.
Use the wash buffer to make 10-fold serial dilutions of each serum sample. Prepare positive and negative controls, according to the manufacturer's instructions. Add 30 microliters of 10-fold serial dilutions of the sample, the positive control, and the negative control to five individual reaction fields on the reagent tray.
Hold the biochip slide by its sides and remove its protective cover without touching its reaction fields. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer. Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the wash buffer, and leave it at the room temperature for 10 minutes.
Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields. Let the slide air-dry for 1 to 2 minutes.
In the dark room, add 25 microliters of fluorescent secondary antibody to each reaction field of the reagent tray. Put the slide on top of the reagent tray, and incubate the setup at room temperature for 30 minutes. Gently rinse the biochip slide with the wash buffer.
Then, immerse it in a 500-milliliter beaker filled with 100 milliliters of the fresh wash buffer, and leave it at room temperature for 10 minutes. Take out the biochip slide from the wash buffer. Use a paper towel to carefully wipe away any remaining buffer from outside of the reaction fields.
Let the slide air-dry for 1 to 2 minutes. Carefully add 1 drop of the embedding medium to each reaction field on the biochip slide. Finally, seal the biochip slide with a coverslip glass, while avoiding air bubbles, and proceed with imaging.
