Immunofluorescence.

Dewaxed paraffin slides were subjected to heat-mediated antigen retrieval in hot 0.01M sodium citrate pH = 6.0 for 20 min before blocking and subsequent antibody labeling. Primary antibody labeling with rabbit anti-Sox2 (Abcam ab97959) 1:500, together with mouse anti-PCNA (Abcam ab29) 1:2,000, or mouse anti-active β-catenin (ABC; Merck 05–665) 1:500 was carried out overnight at 4 °C. Secondary antibody incubation with goat anti-rabbit AlexaFluor-647 (Thermo A-21245) 1:250 and goat anti-mouse AlexaFluor-488 (Thermo A-11-001) 1:250 was carried out for 1 h at room temperature. Slides were counterstained with 1 μg/mL DAPI (Sigma D9542) and mounted in Fluoromount (Sigma F4680). Calretinin (calb2) immunofluorescence was performed with rabbit anti-calretinin (abcam ab702) at 1:1,000. Imaging was carried out on an Olympus BX51 upright epifluorescent microscope and visualized with the software Volocity 6.3.