Immunofluorescence Analysis of Gpr177 in Testes

The control and Gpr177 cKO male mice were killed 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 immunofluorescence, as previously described.^{61 , 64 (link)} In brief, tissue sections were deparaffinised and rehydrated, followed by antigen retrieval in 10 mM sodium citrate buffer. The sections were blocked using a blocking buffer (donkey serum, 0.3% Triton X-100 in PBS) and incubated with primary antibodies against GPR177 (1:200; Santa Cruz, St. Louis, MO, USA, sc-133635), MVH (1:300; Abcam, Cambridge, UK, ab13840) or AQP3 (1:400; kind gift from Dr. Qi Chen) overnight at 4 °C. Sections were washed and incubated with FITC-conjugated secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h and counterstained with DAPI (1:1000; Sigma-Aldrich) to identify the nuclei.