Biotechnology and Bioinformatics (1+1)

Immunostaining

1. Block specimen in Blocking Buffer for 60 minutes.
2. While blocking, prepare primary antibody by diluting to the appropriate dilution in the antibody dilution buffer
3. Aspirate blocking solution, apply diluted primary antibody (eg. Antibody against HPV antigen raised in rabbits).
4. Incubate overnight at 4°C.
5. Rinse three times in PBS for 5 minutes each.
6. Incubate specimen in fluorochrome-conjugated secondary antibody (mouse anti-rabbit) of appropriate dilution in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
7. Rinse in PBS as in step 5.
8. Cover the specimen with antifade reagent
9. Examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.