Sperm Immunocytochemistry for KCNMA1 and KCNU1

Sperm immunocytochemistry was carried out as previously explained [64 (link)]. Briefly, sperm were fixed in 4% paraformaldehyde at room temperature (RT) for 20 min and then centrifuged at 1200× g for 6 min. The pellet was resuspended in 1x PBS (pH = 7.3). Sperm smears were made on poly-D-lysine-coated coverslips and allowed to dry at RT. Dry smears were washed out three times for 5 min each with PBS. Smears were then blocked with 5% BSA in PBS at 4 °C for 2 h. Smears were washed out again with PBS 3 times (5 min for each washout). Primary antibodies, KCNMA1 Rabbit pAb (Cat # A15283, ABclonal, Woburn, MA 01801, USA) and KCNU1 Rabbit pAb (Cat # A14967, ABclonal), were diluted to 1:100 in 5% BSA-PBS, and the washed smears were incubated with the diluted primary antibodies at 4 °C for overnight. After overnight incubation, smears were washed out with PBS 3 times and incubated with the secondary antibody, FITC Goat Anti-Rabbit IgG (H+L) (Cat# AS011, ABclonal), diluted to 1:200 in 5% BSA-PBS for 75 min at RT under dark conditions. Smears were thoroughly washed using PBS and then air-dried at RT before mounting with the permanent mounting reagent. Photographs of the immunolabeled sperm were obtained using confocal microscopy (Leica TCS SP8, Leica Biosystems Nussloch GmbH, Nußloch, Germany).