Whole tubule immunostaining was performed as published with modifications (Gassei & Orwig 2013 (link)). Testes or separated seminiferous tubules were rocked in 4% (v/v) paraformaldehyde or 10% (v/v) neutral buffered formalin at 4 °C overnight. Tubules were washed with PBS (phosphate buffered saline) for 15 min twice, treated with mild dehydration solution (PBS with 10% (v/v) methanol and 0.1% (v/v) TritonX-100) at 4 °C for one hour and blocked with PBSMT (PBS with 1xRoche blocking solution (Roche, Indianapolis, USA) and 0.5% (v/v) TritonX-100) at 4 °C overnight. Tubules were incubated with primary antibody diluted in PMSMT (rabbit anti-POU5F1 1:100, mouse anti-GFP 1:500, rabbit anti-SALL4 1:800 or goat anti-GFRA1 1:50) at 4 °C overnight. Antibody details are in Table S1. Tubules were washed in PBT (PBS with 0.1% (v/v) TritonX-100) six times for 15 min. Secondary antibodies were diluted in PBT with 1 μg/mL DAPI and applied for one hour followed by six 15 min washes in PBT. Tubules were mounted on glass slides in Vectashield or Fluoro-Gel mounting medium. Images were obtained using a Nikon NiE upright microscope equipped with a Hamamatsu Orca-Flash 2.8 sCMOS camera or with a Nikon Eclipse TiS inverted microscope equipped with a Retiga 2000R Fast 1394 camera. Images were acquired with Q-capture Pro software and Image J was used for pseudocoloring and to create overlays of colors.