Alexa fluor conjugated secondaries

For BrdU staining, antigen retrieval (DNA denaturation) was performed with 10mM sodium citrate (pH6) at 95 °C for 20 min, permeabilized in 0.1% Triton X-100 (T8787; Sigma) in PBS (PBST) for 3 × 10 min, then blocked with 20% goat serum (G6767; Sigma) in PBST for 30 to 60 min at room temperature. For P-cadherin staining only, 20% donkey serum (D9663; Sigma) was used instead of goat. Specimens were incubated with primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-RFP (1:500, 600-401-379; Rockland Immunochemical), goat anti-P-cadherin (1:200, AF761; R&D Systems), rabbit anti-non-muscle myosin IIB (1:200, 909901; BioLegend), and rat anti-BrdU (1:200, ab6326; Abcam). After six 1- to 2-h PBST washes, specimens were incubated at 4 °C overnight with Alexa Fluor–conjugated secondaries (Life Technology). Nuclei and F-actin were counterstained with DAPI (4′,6-diamidino-2-phenylindole; 1:5000, 62247; Thermo Fisher Scientific) and Alexa Fluor 488/635 Phalloidin (1:500, A12379, A34054; Invitrogen), respectively. Specimens were washed 6 times with PBST (1 to 2 h per wash) and then mounted on glass slides with 50% glycerol (356352; Calbiochem) in PBS. Z-stacks were acquired by a confocal microscope (TCS SP5; Leica) with oil immersion 40× and 63× objectives.