Immunofluorescence

1. Cover with 3 mL paraformaldehyde fix and incubate for 20 min at room temperature (RT).

2. Wash three times (5 min each) with PBS.

3. Cover with quench and incubate for 10 min at RT (see Note 3).

4. Repeat step 3 two more times (total of three quenches).

5. Wash three times (5 min each) with PBS.

6. Block in 5% serum (in PBS) for 10 min at RT or overnight at 4°C (see Note 4).

7. Incubate with primary antibody for 1 h at RT (see Note 5).

8. Wash three times (10 min each) with PBS.

9. Incubate with secondary antibody 30 min to 2 h at RT in the dark.

10. Wash three times (10 min each) with PBS.

11. Rinse briefly with water.

12. Cover cells with a drop of mounting medium and place a cover slip on top (see Note 6) or mount cover slips on slides if cells were cultured in this manner.

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