Alexa fluor anti mouse or anti rabbit

In this analysis, 1 × 10 4 cells were seeded on sterilized 12-mm-diameter coverslips in 24-well plates and treated as explained above. After treatment, cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Slides were then permeabilized for 10 min with 0.1% Triton X-100 in PBS. Cells were blocked for 30 min with 3% bovine serum albumin, incubated at 4°C overnight with the primary antibody, and then washed and incubated for 40 min with the secondary antibodies. Images were taken with a Leica confocal microscope (Leica, Wetzlar, Germany) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) and the JACoP plug-in to determine colocalization coefficients. The following primary antibodies were used: rabbit anti-FoxO1 (1:500; Abcam, Cambridge, UK), rabbit anti-PDX-1 (1:500; Abcam), rabbit anti-MafA (1:200; Abcam), mouse anti-NeuroD (1:100; Abcam), and rabbit anti-active caspase 3 (1:100; Cell Signaling Technology, Danvers, MA) as a marker of apoptosis. Secondary antibodies were Alexa Fluor antimouse or antirabbit (1:600; Life Technologies, Carlsbad, CA). DNA was stained with DAPI (4′,6-diamidino-2-phenylindole). Results of nuclear localization are expressed as the proportion of DAPI colocalized with each transcription factor.