H 1800

Tissue sections were prepared and subjected to antigen retrieval as described above, then blocked in 0.4% v/v Triton X-100, 1% BSA, and 5% NDS for 60 min followed by overnight incubation in primary antibody prepared in 0.4% Triton X-100/PBS at 4°C. Sections were washed in PBS and incubated with secondary antibodies diluted in 0.4% Triton X-100/PBS for 90 min. Finally, slides were washed 3 times in PBS and coverslipped in DAPI-containing mounting medium (H-1400 or H-1800, Vector Laboratories).

After mice euthanization, the testes were washed in PBS and incubated in 4% paraformaldehyde overnight, followed by five 1-h washes in PBS with shaking at 4°C. Testes were then incubated in 30% w/v sucrose prepared in PBS at 4°C until it sank. Testes are embedded in Tissue-Tek O.C.T. Compound (4583, Sakura), and frozen at −80°C for at least 2 h. Tissue blocks were then sectioned on a cryotome (Leica) to 5 μm thickness and stored at −20°C or −80°C until ready for staining. Sections were briefly hydrated in PBS for 5 min, followed by permeabilization in 0.1% v/v Triton X-100/PBS for 5 min. Sections were then blocked in 3% w/v BSA in PBS with 0.1% v/v Triton X-100 for 1 h at RT, followed by incubation with primary antibody (prepared in blocking buffer) for 1 h at RT. After three washes in PBS, the sections were incubated with secondary antibodies in blocking buffer for 1 h at RT. After three final PBS washes, the slides were mounted in DAPI containing mounting medium (H-1400 or H-1800, Vector Laboratories).

Cell death was assessed in the control and Wnk1^f/f; Wnt7a-Cre testes using the In situ Cell Death Detection Kit (#11684795910, Roche) according to the manufacturer’s instructions. Briefly, formalin fixed paraffin embedded tissues were first sectioned to 5 μm, then deparaffinized in Citrisolv and hydrated through decreased ethanol % (100% X 3, followed by 95%, then 70%, then water). Sections were treated with 20 μg/mL proteinase K (25530-049, Invitrogen) prepared in 10 mM Tris-HCl for 30 min at 37°C. After a brief PBS wash, the sections were incubated with TUNEL reaction mixture (50 μL of enzyme solution mixed into 450 μL Label solution just prior to use) for 60 min at 37°C. Sections were washed 3 X in PBS, followed by coverlipping in DAPI containing mounting medium (H-1800, Vector Laboratories). Note that for each experiment, a negative and positive control were included. For the positive control, the tissue section was incubated with 0.05 U/μL DNase I (18068-015, Invitrogen) for 10 min at RT to artificially induce DNA damage prior to labeling with the TUNEL reaction mixture. For the negative control, the section was incubated with only the Label solution (without the enzyme).