A 21271

Rehydrated sections were subjected to antigen retrieval by microwaving 2 × 8 min at 650 W in citric acid buffer (0.01 M, pH 6) followed by 10 min cooling in citric acid buffer and 20 min washing under cold tap water. Washed slides were incubated 10 min in 3% hydrogen peroxide prior to 10 min washing in PBS supplemented with 0.25% Triton X100 (PBS‐T). Slides were incubated over‐night at 4°C with primary antibody against the pan neuronal marker HuC/HuD (A‐21271, Thermo Fisher Scientific, SE, RRID AB_221448, (Cheng et al., 2016 (link))) diluted 1:400 in PBS with 0.25% BSA. Sections were washed 3 × 10 min in PBS‐T followed by 1 h incubation with SignalStain boost‐detection HRP‐mouse (8125S, Cell Signaling, USA, RRID AB_10547893). Slides were washed 3 × 10 min in PBS‐T followed by development for 1 min with DAB reagent (sk‐4100, Vector, USA, RRID AB_2336382). Slides were washed 10 min under running tap water before being submerged 30 s in hematoxylin for counterstaining. Following counterstaining slides were washed 10 min under running tap water and dehydrated by stepwise washes in increasing grades of ethanol and xylene before being mounted with Pertex™ (Histolab, SE). Slides were scanned on a Nanozoomer 2HT (Hamamatsu, JP) in brightfield mode with 20x objective and analyzed using QuPath software (Bankhead et al., 2017 (link)).

The ROs were fixed in 4% paraformaldehyde at room temperature for 30 min and cryoprotected in 15% sucrose for at least 2 h, followed by 30% sucrose overnight, before being embedded in optimum cutting temperature (OCT) compound. The OCT blocks were sectioned at 10 μm thickness and incubated at room temperature for at least 1 h, before immunostaining or storage at −80 °C. Slides were rinsed in 0.1% Triton X-100 (Sigma-Aldrich) in PBS and blocked with 5% donkey serum in PBS for 2 h, and incubated overnight at 4 °C with primary antibodies diluted in blocking solution. Primary antibodies were used at the following dilutions: HuC/D (1:300; A-21271, Thermo Fisher Scientific), Ki-67 (1:300; ab16667, Abcam, Cambridge, UK), CHX10 (1:100; X1179P, Exalpha Biologicals, Shirley, MA), p-S2448 (1:250; 44-1125G, Invitrogen, Waltham, MA), p-S6 ribosomal protein (1:500; 5346, Cell Signaling Technology, Danvers, MA), AtoH7 (1:500; PA5-35859, Thermo Fisher Scientific). Species-specific secondary antibodies, conjugated with Alexa Fluor 488 or 568, were diluted in antibody dilution buffer and incubated at room temperature for 2 h. Hoechst® 33342 (H1399, Invitrogen) was used for nuclear staining. Fluorescence images were captured using a confocal microscope (DMI8; Leica Camera, Wetzlar, Germany).