Day 1. 

Treat cells with 1X EdU from 1000x stock. . Add 0.2 ul/2 ml complete media.

Day 2. 

1. Wash cells with 1x PBS

2. Fixing: add 500 ul of 4% PFA. Incubate at RT for 15 min.

3. Wash 1x PBS X2

4. Add 0.3% Triton X-100, incubate at RT FOR 10 min.

5. Wash 1x PBS X2

6. Prepare following Rn.
   

1x click IT buffer:               1.7 ml
CuSO4 (Component D):         80 ul
Alexa Fluor (Component B)   5ul
1x ClickIT buffer additive     200ul
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Final vol                                       2 ml 
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7. Add 250 ul/well incubate at RT 30 min.

8. Wash cells with 100 ul of rn. rinse buffer

9. Add DAPI

10. Mount