EdU immunostaining to detect dividing cells

EdU immunostaining to detect dividing cells

1. Dechorionate embryos
2. Place larvae in 1mL of 500uM EdU/5% DMSO in Embryo Medium in 2 mL Tube. 
   1. 10mM EdU/DMSO stock, add 50 ul EdU
   2. 950 ul E3
3. Incubate tube on ice for 1h
4. Replace EdU with 10 mM EdU/H2O stock, transfer larvae to 12-well plate (n=10/well) and back to 28.5C. Fix in 4% PFA when desired, then keep in PBST at 4C.
5. Rinse in dH2O, place in Acetone at -20C for 7min, rinse in dH2O (no need for proK).
6. Permeabilize for 1h in PBS/1%DMSO/1%Triton.
7. Prepare 1X Click-iT EdU buffer additive fresh (dilute 10x stock with dH2O).
8. Prepare Click-iT reaction cocktail (360uL 1x Reaction buffer + 16uL CuSO4 solution + 1uL Alexa Fluor azide + 40uL Reaction buffer additive).
9. Use reaction cocktail within 15min of preparation. Switch embryos to reaction cocktail, incubate for 1h in dark at room T.
10. Remove Reaction cocktail and rinse embryos in PBT 5x5min rinses. 
11. If performing antibody staining, block at this step, and proceed as with usual immunostaining protocol.