BP-pacFA protocol version 2

1. incubate with 10μM BP-pacFA in HBSS for 10min at 37°C
2. expose to 525nm light for 2min to uncage the probe
3. return to the incubator for varying amounts of time to allow for metabolism
4. expose to 365nm light for 60s to cross-link on ice, and immediately replaced with ice-cold methanol (-20°C) for 20min on ice
5. permeabilized in 0.2% Tween-20 in PBS for 20min at RT
6. wash three times with PBS for 5min each time
7. incubate with 50 μL click mix for 1h in the dark
8. wash three times with PBS for 5min each time
9. blocked with PBS supplemented with 3% BSA and 0.02% Triton X-100 for 1h in the dark
10. incubate with primary antibody in PBS supplemented with 3% BSA and 0.02% Triton X-100, overnight in the dark at 4°C
11. wash three times with PBS for 5min each time
12. incubate with second antibody in PBS for 2h in the dark at RT
13. wash three times with PBS for 5min each time
14. incubate with DAPI
15. wash three times with PBS for 5min each time
16. 制片