Tissue Fixation and Immunostaining Protocol for Testis Analysis

The control and Gata4 cKO male mice were euthanized via cervical dislocation and the testes were immediately fixed in Bouin's solution for hematoxylin and eosin (H&E) staining or in 4% formaldehyde (PFA) in PBS for immunostaining, as previously described [57 (link)-59 (link)]. In brief, tissue sections were deparaffinized and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. For immunohistochemistry (IHC), the sections were blocked with 5% bovine serum albumin (BSA) and incubated with the primary antibody at 4°C overnight, and then the secondary antibody was applied for 1 hour. Staining was visualized using a DAB substrate kit (Zhongshan Technology, Beijing, China). For immunofluorescence (IF), the sections were blocked using a blocking buffer (donkey serum, 0.3% Triton X-100 in PBS) and incubated with primary antibodies overnight at 4°C. Sections were washed and incubated with FITC or TRITC-conjugated secondary antibodies (Jackson ImmunoResearch, CA, USA) for 1 hour and counterstained with DAPI (Sigma, MO, USA) to identify the nuclei. The primary and secondary antibodies used for immunostaining are listed in Table S2.