Mouse anti rat pcna

Immunofluorescence (IF) was performed on free-floating 60 μm sections as previously described (Fontaine et al. 2013) (link). For BrdU IF and proliferating cell nuclear antigen (PCNA) IF, epitope retrieval was achieved using either 2 M HCl (in PBS with 0.1% Tween) for 1 h at 37°C or HistoVT One (Nacalai Tesque, Japan) for 20 min at 90°C. Then, a blocking step of 1 h was followed by primary antibody incubation: rat anti-BrdU (1:250; Abcam), mouse anti-ratPCNA (1:100; Santa Cruz Biotechnology) or custommade polyclonal rabbit anti-medakaLhβ (1:2000 (1: (Burow et al. 2018)) ). For goat anti-human Sox2 (1:500; Immune Systems, UK), blocking was prepared according to supplier recommendations. Amplification was performed using AlexaFluor 555 or 647 secondary antibodies (1:1000; Invitrogen). Nuclei in Lhβ-IF and Sox2-labeled tissues were stained with DAPI (1:1000; 4′,6-diamidino-2-phenylindole dihydrochloride; Sigma). Control experiments were performed using the same protocol without primary antibody.