Immunohistochemical Staining of Drosophila Testes

Immunohistochemical staining was performed as described previously with a few modifications (Lin et al., 2021 (link)). Drosophila adult testes were dissected in 1 × phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde for 20 min. After washing with PBST (0.3% Triton in 1× PBS) three times, the testes were incubated in a blocking buffer (5% goat serum in 0.1% PBST) for 1 h. Next, the blocking buffer was replaced by primary antibodies mixed in PAT (1% BSA, 0.1% Triton and 0.01% sodium azide in 1× PBS) and the incubation lasted for overnight. Then, the testes were washed three times with 0.1% PBST and incubated in secondary antibodies mixed in 0.1% PBST for 1 h. After washing with 0.1% PBST three times, the tissues were stained with DAPI for 10 min and washed three times before mounting with Fluoromount-G. DAPI (#MBD0015, Sigma-Aldrich, Germany), DCP1 (#9578S, Cell signaling, USA).