HeLa Cell Immunostaining

1. The day before, cells need to be split. I usually wait until they are ~80% confluent, and then split them into chambered coverglass wells 50uL cells: 450uL media. Be sure to spin off trypsin so they adhere better.

2. Allow to grow overnight in 37C CO2 incubator, about 18-26 hours.

3. Remove chamber from incubator and check cells on scope to make sure they are adhered and look healthy.

4. Carefully aspirate off media from corner of well (avoid aspirating off too many cells).

5. Immediately add 500uL pre-extraction buffer (0.2% Triton X-100, 100mM Pipes pH 6.8, 1mM MgCl2, 5mM EGTA), incubate 1 min @ RT.

6. Carefully aspirate off buffer, add 200uL fixative (3.2% Paraformaldehyde in PBS), incubate 10min @ RT.

7. Aspirate off fixative, wash cells 1X in 1X PBS. Block with 200uL 3% BSA (30mg/mL in PBS) for 1 hour @ RT.

8. Aspirate off block, add ~100uL of primary Ab (3% BSA, 0.1% TX-100, Bub1 1:500, DM1A 1:1000, & RNase A .08mg/mL in PBS). Incubate chamber (wrapped in parafilm) overnight @4C.

9. The next morning, wash cells 3X in PBS+0.01% TX-100 for 5min @ RT.

10. Add secondary AB (goat a-mouse 488 1:300, goat a-rabbit 594 1:300, Draq5 1:1000, 3% BSA, 0.1% TX-100 in PBS) for 1hr @ RT in the dark.

11. Wash cells 3X in PBS+0.01% TX-100 for 5min @ RT.

12. Mount with 20uL mounting media (1X PBS, 70% glycerol).

13. If not imaging immediately, store chamber @4C wrapped in parafilm.