Immunofluorescence in situ for EdU and BrU incorporation.

Cells grown on coverslips were fixed in 2% formaldehyde in PBS, permeabilized in 0.5% Triton X-100 in PBS, and incubated in a Click-It reaction mix as described previously (65). Alexa Fluor 488-azide (Click It Chemistry Tools) was used in Click-It reactions for EdU incorporation analyses, and biotin-azide (Click It Chemistry Tools) was used for PLAs and for EdU-BrU costaining. In some PLAs, a mixture of Alexa Fluor 488 and biotin-azide was used at a 40:1 molar ratio, respectively. For BrU-EdU costaining after Click-It reactions with biotin-azide, cells were blocked in PBS–3% bovine serum albumin (BSA)–3% normal goat serum and then incubated with anti-BrdU (GeneTex) and antibiotin (Vector Laboratories) antibodies, followed by Alexa fluorophore-conjugated secondary antibodies (Invitrogen). Note that both RFP and GFP signals, if still expressed in the cell lines analyzed, are too low to affect the immunofluorescence (IF) signal; in addition, GFP fluorescence is inhibited by the click reaction.