Rat anti vasa

Fly testes were dissected in 1× phosphate-buffered saline (PBS), fixed for 30 min in 4% paraformaldehyde, washed with 0.3% PBS-Triton X-100 (PBST) three times, and blocked in 5% bovine serum albumin for 30 min. Testes were incubated with primary antibodies at room temperature for 1 h. Then, testes were washed three times in 0.3% PBST and incubated with secondary antibodies at room temperature for 1 h in the dark. After washing three times again with 0.3% PBST, testes were stained with Hoechst-33342 (1.0 mg/ml, C0031, Solarbio, Beijing, China) for 5 min before mounting. The primary antibodies used were as follows: rat anti-Vasa [Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Dept of Biology, Iowa City, IA, USA; 1:20], mouse anti-1B1 (DSHB, 1:50), mouse anti-FasIII (DSHB, 1:50), rat anti-Zfh1 (a gift from Prof. Chao Tong, 1:1000), mouse anti-Eya (DSHB, 1:30), rabbit anti-PH3 [#53348, Cell Signaling Technology (CST), Danvers, MA, USA; 1:1000], rabbit anti-dpERK (#4370, CST, 1:200). Secondary antibodies conjugated with A488, Cy3, or A647 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were diluted at 1:1000.

Adult ovaries were dissected in Grace's medium and fixed in 4% (vol/vol) formaldehyde for 10 min. The ovaries were washed with PBT (1X PBS, 0.5% BSA, and 0.3% Triton-X 100) and stained with primary antibody overnight at 4°C. The ovaries were washed and incubated in secondary antibody at room temperature for 5 hrs. Ovaries were then washed again and mounted in Vectashield containing DAPI (Vector Laboratories). The following primary antibodies were used: mouse anti-Hts (1B1) (1∶20), mouse anti-Engrailed (4D9) (1∶2), mouse anti-Broad-core (25E9.D7) (1∶10), mouse anti-Rho1 (P1D9) (1∶50) and rat anti-VASA (1∶20) (Developmental Studies Hybridoma Bank), mouse anti-β-galactosidase (1∶200) (Promega), rabbit anti-APC1 [54] (link) (1∶1000)(gift of E. Wieschaus), Rabbit anti-α-Spectrin [55] (link)(1∶1000)(gift from Ron Dubreuil), rabbit anti-GFP (1∶1000) (Molecular Probes), rat anti-HA 3F10 (Roche) and rabbit anti-cleaved Caspase-3 (1∶250) (Cell Signaling Technology). Fluorescence-conjugated secondary antibodies (Jackson Laboratories) were used at a dilution of 1∶200.

Testes were dissected in PBS, fixed in 4% formaldehyde for 20mins and washed by PBSTw (PBS with 0.1% Tween-20) for 3 times. Permeabilization of testes was done by incubation with PBST (PBS with 0.5% Triton-X) for 30mins. Testes were then blocked by 5% BSA in PBSTw for at least an hour, before incubation with primary antibody in 5% BSA in PBSTw at 4°C overnight. Testes were washed 3 times with PBSTw and incubated with secondary antibody in 5% BSA in PBSTw at room temperature for 2hrs, followed by another 3 washes with PBSTw. Before mounting in VECTA-SHIELD, testes were stained by DAPI (1:5000) for 10mins and rinsed once with PBS. The following primary antibodies were used: mouse anti-Fas3 (7G10, 1:200), mouse anti-γH2Av (UNC93-5.2.1, 1:400) and rat anti-Vasa (concentrated, 1:100) were obtained from Developmental Studies Hybridoma Bank.

Immunofluorescent staining was performed as described previously [47 ]. Briefly, testes were dissected in phosphate-buffered saline (PBS) and fixed in 4% formaldehyde in PBS for 30 to 60 minutes. Next, testes were washed in PBST (PBS + 0.3% TritonX-100) for at least 30 minutes, followed by incubation with primary antibodies in 3% bovine serum albumin (BSA) in PBST at 4°C overnight. Samples were washed for 60 minutes (3 times for 20 minutes each) in PBST, incubated with secondary antibodies in 3% BSA in PBST at 4°C overnight, and then washed for 60 minutes (3 times for 20 minutes each) in PBST. Samples were then mounted using VECTASHIELD with 4,6-diamidino-2-phenylindole (DAPI) (Vector Lab, H-1200).
The primary antibodies used were as follows: rat anti-Vasa (1:20) and mouse anti-Bam (1:20) were obtained from the Developmental Studies Hybridoma Bank (DSHB); Rabbit anti-Smad3 (phospho S423 + S425) (1:100, Abcam, ab52903); Guinea pig anti-STAT92E (1:2000, a gift from Yukiko M. Yamashita); rabbit anti-Zfh1 (1:4000; a gift from Ruth Lehmann).
AlexaFluor-conjugated secondary antibodies were used at a dilution of 1:400.
Images were taken using a Zeiss LSM800 confocal microscope with a 63× oil immersion objective (NA = 1.4) and processed using Image J and Adobe Photoshop software.