Dmi4000 confocal microscope

For immunohistochemistry on frozen sections, testes were fixed in 4% paraformaldehyde in PBS at 4 ^oC overnight, dehydrated through a sucrose gradient, embedded in OCT and cryosectioned at 10 µm increments. Testes collected after birth were embedded in paraffin wax using the following methods: samples were fixed in 4% paraformaldehyde in PBS at 4 ^oC overnight, dehydrated through an ethanol gradient, and embedded in paraffin wax. 6-µm-thick sections were dewaxed and rehydrated in a series of alcohol just prior to immunostaining. All tissue sections (frozen or paraffin sectioned) were preincubated with 5% normal donkey serum in PBS for 1 hour, then incubated with either anti-AMH (1:500; sc-6886, Santa Cruz), anti-GFP (1:500; A1172, Life Technologies) or anti-TRA98 (1:1000, MBL international), in PBS-Triton X-100 solution with 5% normal donkey serum at 4 ^oC overnight. The antibody-labeled tissue sections were then washed 3 times in PBS and incubated in the appropriate secondary antibody (1:500; Invitrogen) before a final 3 washes (with PBS) and mounting in Vector Mount with DAPI (Vector Labs). Slides were imaged under a Leica DMI4000 confocal microscope.

Images were captured on a Leica DMI6000B Microscope and processed with LAS AF computer software. All images were captured at 5X magnification over target regions, with a total of 4–6 images taken bilaterally in adjacent sections. Within each region and each cohort a constant gain, exposure, and light intensity were used for all images. Images for insets were captured using a Leica DMI4000 confocal microscope. All confocal images were captured with a 40X objective lens and within each region constant imaging parameters were used for all images.