Anti sycp3 ab

IF of whole-mount seminiferous tubules stretched on slide glass was carried out as previous reported (Nakagawa et al., 2010 (link)) using anti-GFP Ab (Nacalai Tesque; 1:400), anti-GFRα1 Ab (R&D Systems; 1:400), anti-Rarγ Ab (Cell Signaling; 1:200), anti-Plzf Ab (Santa Cruz; 1:200), anti-KIT Ab (R&D systems; 1:500), and anti-SYCP3 Ab (Abcam; 1:500). Paraffin sections (7 μm-thick) of the testes were immunostained using anti-GFP Ab (Thermo Fisher; 1:300 or Abcam; 1:300), anti-Plzf Ab (EMD Millipore; 1:100), anti-SYCP3 Ab (Abcam; 1:500) and anti-Collagen IV (Abcam; 1:500) after antigen retrieval in 0.01 M citrate (pH 6.0; > 90C maintained for 10 min). All secondary antibodies were Alexa Fluor-conjugated, purchased from Thermo Fisher Scientific, and used at 1:400 dilutions. Slides were mounted in Fluoro-KEEPER Antifade Reagent (Nacalai Tesque). Observations and scoring were carried out using an Olympus BX51 upright fluorescence microscope equipped with a DP72 CCD camera, an Olympus FLUOVIEW FV3000 confocal laser scanning microscope, or a Leica TCS SP8 confocal system.