Neuronal apoptosis was analyzed as previously described (Abramowski et al., 2018 (link)). E15.5 fetal heads were fixed overnight at 4°C by immersion in 4% paraformaldehyde and embedded in paraffin with a Tissu-tek processor (VIP, Leica). 5 μm coronal sections were then obtained using a microtome (Leica RM2125RT) and mounted onto glass slides for histological analyses. After paraffin removal and citrate treatment, the brain sections were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 15 min and incubated for 2 hr with 7.5% fetal bovine serum and 7.5% goat serum in PBS. The sections were incubated with rabbit anti-CC3 (Cell Signaling 9661) overnight at 4°C. After washing, the sections were incubated with goat anti-rabbit Alexa Fluor 488 or 594 conjugated secondary antibody (ThermoFisher) for 1 hr. After washing, nuclear staining was achieved by incubation with 4′-6-diamidino-2-phenylindole (DAPI) to quantify apoptosis induction by the detection of pyknotic nuclei (Roque et al., 2012 (link)). Slides were mounted under Fluoromount (Southern Biotechnologies Associates). Tissues were examined under a fluorescence microscope (50i, Nikon, Japan) with a 10× (NA = 0.3) objective in three channels (appearing red, green, and gray) as separate files. These images were then stacked with Photoshop software (Adobe).
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