Immunofluorescence Analysis of Apoptosis in hPSCs

hPSCs were analyzed for in situ immunofluorescence^{5 (link),56 (link)}. Briefly, cells were rinsed with ice-cold PBS and fixed in PBSA (PBS with 0.1% bovine serum albumin) with 4% formaldehyde for 45 min. After two washes cells were permeabilized with 0.1% Triton X-100 in PBSA with 10% normal goat serum for 30 min, washed twice, and stained with a rabbit polyclonal antibody anti-active CASPASE-3 (ab13847, Abcam Inc., Cambridge, MA, USA). Fluorescent secondary antibody Alexa Fluor 488-conjugated anti-rabbit IgG (Thermo Scientific) was used to localize the antigen/primary antibody complexes. Cells were counterstained with DAPI and examined under a Nikon Eclipse TE2000-S inverted microscope equipped with a 20X E-Plan objective and a super high-pressure mercury lamp. The images were acquired with a Nikon DXN1200F digital camera controlled by the EclipseNet software (version 1.20.0 build 61).