Quantifying Apoptosis in Newborn and Fetal Mice

For analysis of cell death in newborn mice, slides were scanned using a Hamamatsu Nanozoomer (Hamamatsu Photonics K.K.) and AC3+ cell quantification was performed using Aperio Image Scope (Leica Biosystems Inc). For the smaller experiments in which cell death was examined in fetuses, AC3 immunostaining was quantified live on a microscope using Stereo Investigator software (MBF Biosciences). In both cases, we drew contours around the areas of interest and AC3+ cells within each contour were quantified as previously described (Ahern et al., 2013 (link); Mosley et al., 2017 (link); Castillo-Ruiz et al., 2018a (link),b (link)). Because AC3 cells are relatively sparse, all positive cells could be counted and random sampling was not required. Cells were counted only when the cell body was clearly visible within the section. The sum of AC3+ cell counts across all sections was divided by total area sampled, and then multiplied by section thickness to obtain the density of AC3+ cells per mm³ for each animal. Investigators blind to experimental conditions performed all analyses.