The reproductive systems of male D. melanogaster were dissected in Grace’s Insect Medium, supplemented (Thermo Fisher Scientific), and fixed in 4% paraformaldehyde in Grace’s Insect Medium for 30 to 60 min at room temperature (RT). The fixed samples were washed three times in PBS supplemented with 0.1% Triton X-100. After washing, the samples were transferred to blocking solution (PBS with 0.1% Triton X-100 and 2% BSA; MilliporeSigma; A3608) for 1 h at RT and incubated with a primary antibody in the blocking solution at 4 °C overnight. The primary antibodies used in this study were mouse anti-GFP monoclonal antibody (clone GFP-20; MilliporeSigma; G6539; 1:1,000) and rabbit anti-Tdc2 antibody (Abcam; ab128225; 1:2,000) (48 (link)). After washing, Fluorophore (Alexa Fluor 488 and 555)-conjugated secondary antibodies (Thermo Fisher Scientific; A11001 and A32732; 1:200) were applied in blocking solution, and the tissue was incubated for 2 h at RT. After a final wash, all samples were mounted in FluorSave reagent (MilliporeSigma). Samples were visualized on a Zeiss LSM 700 confocal microscope. Images were processed using the ImageJ package (49 (link)).