Nie upright microscope

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.

Images for all immunofluorescence experiments (except Supplementary Figure S1H) were acquired using the Leica SP8 scanning confocal system with the DMi8 inverted microscope. Leica LASX software (Leica Microsystems) was used for image acquisition. All the images were taken using 63X, 1.4NA oil immersion objective at RT. Images in Supplementary Figure S1H were obtained using Nikon NiE upright microscope with Hamamatsu Orca-Flash 2.8 sCMos high resolution camera. Following image acquisition, images were processed using Image J (National Institutes of Health, Bethesda, MD). For quantifying the percentage of cells with colocalization, at least 100 cells were scored. Each experiment was performed in 3 biological replicates. Colocalization was also confirmed using RGB profiler plugin on ImageJ as described previously (19 (link)).