Sc 17320

Cerebella of mice were formalin-fixed and paraffin-embedded (8 μm sections; see above). Alternatively, brains were fixed with 4% PFA in PBS for 2.5 h on ice, followed by cryoprotection with 30% sucrose in PBS overnight. Fixed tissues were embedded in O.C.T Compound (Sakura), frozen on dry ice and cryopreserved at −80 °C for cryosections (12 μm thickness). For staining with an anti-PCNA antibody, sections were subjected to heat antigen retrieval with 10 mM citrate buffer for 30 min. After blocking with 10% normal donkey serum in PBS-T (0.1% Triton X-100 in PBS) for 30 min at room temperature, sections were incubated with primary antibody overnight at 4 °C. Incubation with secondary antibody was performed for 30 min at room temperature. Subsequently, sections were mounted using the ProLong Gold antifade reagent (Lifetechnologies). The antibodies used for IHC were anti-Pax6 (1:1,000, Covance, PRB-278P), anti-PCNA (1:1,000, Merck Millipore, NA03), anti-GFP (1:1,000, Abcam, ab13970), anti-GFP (1:1,000, Cell Signaling, 2,555), anti-Pax2 (1:1,000, Invitrogen, 71-6000), anti-Calbindin (1:1,000, Millipore, AB1778), anti-Sox2 (1:500, Santa Cruz, sc-17320), anti-p27 (1:1,000, BD Biosciences, 610242) and anti-Atoh1 (1:1,000, Muguruma et al.42 (link)).