Rabbit anti active caspase 3

Human umbilical vein endothelial cell cultured in 8-well chamber glass slides coated with BME (Trevigen Inc., USA), as described previously (13 (link)), were treated for 5 min with mixture containing 0.1% Triton X-100 and 4% PFA containing 5% sucrose, and fixed for an additional 25 min with 4% PFA containing 5% sucrose. The cells were washed for 10 min with PBS and an additional 15 min with PBS containing 0.05% Tween 20 (PBS-T). Next, fixed cells were blocked with IF buffer (130 mM NaCl, 7 mM Na₂HPO₄, 3.5 mM NaH₂PO₄, 7.7 mM NaN₃, 0.1% BSA, 0.2% Triton X-100, and 0.05% Tween 20) containing 10% donkey serum for 1 h followed by overnight incubation at 4°C with rabbit anti-active-caspase-3 (1:400) (Cat # 559565; BD Biosciences). The cells were washed three times with PBS for 15 min each and incubated for 1 h with donkey anti-rabbit conjugated to Alexa Fluor^® 647 (Invitrogen, USA) at room temperature. Next, the cells were washed as mentioned earlier and mounted with VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). Immunofluorescent images were captured by Zeiss LSM 700 confocal laser scanning microscope (magnification 40×).
For F-actin staining, cells were incubated overnight with Alexa Fluor 488 Phalloidin (1:40) (Molecular Probes, USA), washed three times with PBS for 15 min each and mounted with VECTASHIELD mounting medium with DAPI.