Fig3c pHH3 and TUNEL co-staining in Wdr62 genetrap adult testis

Raw image dataset for pHH3 and TUNEL co-staining on adult Wdr62 genetrap testis sections. <br>pHH3 staining was performed prior to TUNEL. Please see Fig3a and 3b. <br>1) Dewax slides2) Antigen retrieval: 10mM Sodium citrate pH6, 95C 15 mins.3) Blocking (20% FBS, 0.2% Triton X in PBS) 1hr RT.4) Primary antibody (rabbit anti-pHH3 (Millipore 06-570) 1:400 diluted in blocking buffer, 4C overnight. 5) Secondary antibody (goat anti-rabbit Alexa 488 (Invitrogen A11034) 1:250 diluted in blocking buffer, RT for 1hr.6) Take 100uL out from labelling reagent #2 for negative control. Then add reagent #1 (50uL) into the rest of the labelling reagent #2 (450uL). Incubate at 37C for 1hr. 7) Counterstain with DAPI 5mg/ml (Sigma D3817) 1:10,000 in PBS for 5 mins RT. 8) Mount in Prolong Diamond (Thermo Fisher Scientific). 9) Images were captured using Leica SP8 inverted confocal microscope. <br><br>

针对成年Wdr62基因陷阱（gene trap）小鼠睾丸组织切片的磷酸化组蛋白H3（phospho-histone H3, pHH3）与末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（Terminal deoxynucleotidyl transferase dUTP nick end labeling, TUNEL）共染色原始图像数据集。实验先进行pHH3染色，再开展TUNEL染色，具体详见图3a与图3b。
1) 切片脱蜡处理
2) 抗原修复：采用pH 6的10mM柠檬酸钠缓冲液，95℃孵育15分钟
3) 封闭：使用含20%胎牛血清（fetal bovine serum, FBS）、0.2% Triton X-100的磷酸盐缓冲液（phosphate buffered saline, PBS），室温孵育1小时
4) 一抗孵育：将兔抗pHH3抗体（默克Millipore 06-570）以1:400的比例稀释于封闭缓冲液中，4℃孵育过夜
5) 二抗孵育：将山羊抗兔Alexa Fluor 488（赛默飞世尔科技Invitrogen A11034）以1:250的比例稀释于封闭缓冲液中，室温孵育1小时
6) 阴性对照制备：从标记试剂2中吸取100μL作为阴性对照；剩余标记试剂2（450μL）中加入试剂1（50μL），37℃孵育1小时
7) 复染：采用以PBS稀释至1:10000浓度的5mg/mL 4',6-二脒基-2-苯基吲哚（4',6-diamidino-2-phenylindole, DAPI）染液（西格玛Sigma D3817），室温孵育5分钟
8) 封片：使用ProLong Diamond抗荧光淬灭封片剂（赛默飞世尔科技Thermo Fisher Scientific）进行封片
9) 图像采集：采用徕卡SP8倒置激光共聚焦显微镜完成图像拍摄