Mouse neurocult nsc proliferation medium

The forebrain of each mouse embryo at E13.5 was dissected and mechanically dissociated. Cells from each forebrain were seeded into a 100 mm Petri dish at a density of 2 × 10⁵ cells/ml in 10 ml of mouse NeuroCult NSC Proliferation Medium (Stem Cell Technologies) supplemented with epidermal growth factor (20 ng/ml; Gibco) and basic fibroblast growth factor (10 ng/ml; Gibco) for selective neurosphere growth. Neurospheres were passaged when they reached 100–150 μm in diameter. For immunochemistry, mouse NPCs were grown in 35 mm glass-bottom dishes (MatTek) at a density of 8 × 10⁴ cells/ml for 24 h, fixed using 4% PFA, and permeabilized with 0.4% Triton-X in PBS. Subsequently, they were incubated overnight at 4 °C with primary antibodies including mouse anti-Ki67 (1:200; Cell Signaling) and chicken anti-Nestin (1:500; Novus) for the identification of proliferating NPCs. This was followed by incubation with secondary antibodies: goat anti-mouse IgG (conjugated with Alexa Flour 488; Invitrogen) and goat anti-chicken IgG (conjugated with Alexa Fluor 568; Invitrogen). Nuclei were counter-stained with DAPI. Immunostaining was examined by a Zeiss META 710 confocal microscope and images were imported into Image-ProPlus, version 7.0 for qualification.