C0031

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.

A percentage of 4–5% isoflurane was used to induce anesthesia, and 1%–3% isoflurane was applied to keep the mice anesthesia in the whole operated process. The left and right epididymis were removed respectively, and then the caudal epididymis was excised and transferred to a dish containing 2 mL 37 °C prewarmed Enriched Krebs-Ringer bicarbonate medium (EKRB, G0430, Solarbio Science & Technology, Beijing, China) [17 (link),18 (link),19 (link)] with 2% FBS, and immediately cut into 4 pieces to fully release the mature sperms into the medium. After 10 min, the sperm suspension was filtered through 40 μm diameter strainers to remove small clumps and then evenly and gently transferred and stained with Hoechst 33,342 (5 μg/mL, C0031, Solarbio Science & Technology, Beijing, China) for 45 min at 35 °C in the dark, gently mixing to keep the sperm equalization every 15 min.