Immunofluorescence Staining of Gonadal Tissues

Antibody staining of dissected gonads was carried out essentially as described (Lee et al. 2006 (link)). Briefly, dissected gonads were fixed and permeabilized in 1 mL of pre-chilled 100% methanol at −20°C for 10 min. Gonads were then immediately pelleted by centrifugation at 376 RCF for 2 min and treated with 1 mL of pre-chilled 100% acetone at −20°C for 10 min. Samples were washed twice with 1 mL of PBST 0.01% (1× concentrated PBS plus 0.01% vol/vol Triton X-100) and blocked in 1 mL of PBST 0.1% (1× PBS plus 0.1% vol/vol Triton X-100) plus 3% wt/vol BSA for 30 min at room temperature. Samples were then incubated at 4°C overnight with monoclonal anti-FLAG M2 antibody produced in mouse (Sigma) at a concentration of 1:1000 in 100 µL of PBST (PBS plus 0.1% Triton X-100 plus 3% BSA). Samples were washed three times for 5 min with 1 mL of PBST (0.1% Triton X-100) and then incubated for 2 h at room temperature with Alexa 488 conjugated secondary antibody (Jackson ImmunoResearch) at a concentration of 1:500 in 100 µL of PBST (0.1% Triton X-100 plus 3% BSA). 4′,6-diamidino-2-phenylindole (DAPI) (0.5 ng/μL) was included to visualize DNA. Confocal images were taken using a Leica TCS SP8. Antibody staining of dissected gonads for phospho-histone H3 (a marker of actively dividing cells) was carried out as described (Seidel and Kimble 2015 (link)).