Immunocytochemistry protocols (ICC protocols)

Specimen preparation

1. Coat coverslips with poly-L-lysine for 5 min at room temperature.

2. Aspirate liquid, allow coverslips to dry completely and sterilize them under UV light for at least 30 min.

3. Grow cells on glass coverslips .Note:If the specimen is suspension cell , cytospin or smear preparation is needed.

Immunostaining

1. Rinse coverslips two times with PBS.

2. Fix cells with 4% paraformaldehye in PBS for 15 min at room temperature.

Note: Paraformaldehye is toxic, use only in fume hood.

3. Aspirate fixative, rinse two times in PBS for 5 min each.

4. Permeabilize cells with 0.1-0.5% triton x-100 in PBS for 10 min.

Note: Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. These include intracellular proteins and transmembrane proteins whose epitopes are in the cytoplasmic region.

5. Aspirate triton x-100, rinse two times in PBS for 5 min each.

6. Incubate cells in 10% normal goat serum in PBS for 30 min at room temperature.

7. Aspirate goat serum, incubate sections with primary antibody at appropriate dilution in PBS overnight at 4°C or 1 hour at 37°C.

8. Rinse three times in PBS for 5 min each.

9. Incubate cells with fluorochrome-conjugated secondary antibody at appropriate dilution in PBS for 1 hour at 37℃ in dark.

10. Rinse three times in PBS for 5 min each in dark.

11. Incubate cells with 1 μg/ml DAPI.

12. Mount coverslip with a drop of mounting medium.