Mouse anti 1b1

Fly testes were dissected in 1x phosphate-buffered saline (PBS) and fixed for 30 min in 4% paraformaldehyde. After washing three times in 1x PBS with 0.1% Triton X-100 (PBST) and blocking for 1 h in 5% bovine serum albumin, the testes were incubated with primary antibodies for 1 h at room temperature. Then, the samples were washed three times for 10 min in 0.1% PBST and incubated for 1 h with secondary antibodies at room temperature followed by the final three washes in 0.1% PBST. Testes were then stained with Hoechst 33342 (1.0 mg/mL; Invitrogen, CA, USA) for 5 min before mounting.
The primary antibodies used in this study were as follows: rabbit anti-Vasa (1 : 200; Santa Cruz Biotechnology, TX, USA), mouse anti-1B1 (1 : 50; Developmental Studies Hybridoma Bank, IA, USA), rat anti-DE-cadherin (1 : 20; DSHB), mouse anti-Eya (1 : 50; DSHB), rat anti-Zfh1 (1 : 1000; a gift from C Tong; Life Sciences Institute, Zhejiang University, Zhejiang, China), rabbit anti-PH3 (1 : 400; Cell Signaling Technology, Leiden, Netherlands), and mouse anti-FasIII (1 : 50; DSHB). Secondary antibodies were conjugated to A488, Cy3, or A647 (Molecular Probes and Jackson Immunological) and used at a dilution of 1 : 1000.

Immunofluorescence was performed as previously described (Hong et al., 2003 (link); Iida and Lilly, 2004 (link)). Primary antibodies used were as follows: rabbit anti-GFP (Invitrogen, 1:1000); rabbit anti-γ−H2Av (Active Motif, 1:500); rabbit anti-C(3)G 1:3000 (Hong et al., 2003 (link)); mouse anti-1B1 (Developmental Studies Hybridoma Bank, 1:100); mouse anti-γ-H2Av (Developmental Studies Hybridoma Bank, 1:5000); mouse anti-C(3)G (kindly provided by R. Scott Hawley, 1:200) (Page and Hawley, 2001 (link)). Alexa-488 and Alex-594 (Invitrogen, 1:1000) secondary antibodies were used for fluorescence. After staining, ovaries were mounted in prolong gold antifade reagent with DAPI (Life Technology). Images were acquired on either a Leica SP5 confocal microscope or Zeiss LSM 880 with Airyscan confocal microscope.

Mouse anti-V5 (1:400, Sigma-Aldrich V8012), mouse anti-1B1 (1:1, Developmental Studies Hybridoma Bank), goat anti-Vasa, mouse anti-Orb (1:20, Developmental Studies Hybridoma Bank), rabbit anti-Miranda (1:100), guinea pig anti-Miranda (1:200), rabbit anti-n-terminal lola (Giniger et al. 1994 (link)), mouse anti-HA (1:50), and rat anti-ELAV (1:50, Developmental Studies Hybridoma Bank).