Immunofluorescent Staining for Proliferation Markers

To visualize BrdU or PCNA, antigen retrieval was first performed using sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) as previously described [38 (link),40 (link),45 (link)]. Sections were then incubated in the primary antibody (Rat anti-BrdU [BU1/75(ICR1)], Abcam, ab6326, Cambridge, United Kingdom and Accurate Chemical and Scientific, OBT0030, Westbury, NY; Mouse anti-PCNA (clone PC 10), P8825, Sigma Aldrich; Mouse anti-HuC/D (16A11), A21271, Invitrogen, Carlsbad, CA) overnight at 4°C. After washing with PBST, sections were incubated in secondary antibodies (Alexa Fluor-conjugated 488, 594, and 647 goat anti-primary, Thermo Fisher Scientific) for 1 hour at room temperature. Nuclei were stained with either DAPI (Thermo Fisher Scientific) or TO-PRO-3 (Thermo Fisher Scientific). Fluorescence images were captured using a Leica TCS SP5 or SP8 confocal microscope (Leica Microsystems, Werzler, Germany). To visualize EdU-labelled cells, a Click-iT Assay kit (Thermo Fisher Scientific) was used.