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>

本数据集为成年Wdr62基因陷阱睾丸切片的磷酸化组蛋白H3（pHH3）与原位末端脱氧核苷酸转移酶介导的dUTP缺口末端标记染色（TUNEL）共染色原始图像数据集。染色顺序为先进行pHH3染色，再进行TUNEL染色，具体参见图3a与图3b。
1) 脱蜡处理切片
2) 抗原修复：采用10mM柠檬酸钠缓冲液（pH 6），95℃孵育15分钟
3) 封闭：使用含20%胎牛血清（FBS）、0.2% Triton X的磷酸盐缓冲液（PBS），室温（RT）孵育1小时
4) 一抗孵育：以封闭液稀释兔抗pHH3一抗（Millipore 06-570），稀释比例1:400，4℃孵育过夜
5) 二抗孵育：以封闭液稀释山羊抗兔Alexa 488二抗（Invitrogen A11034），稀释比例1:250，室温孵育1小时
6) 阴性对照制备：取100μL标记试剂#2作为阴性对照；将50μL试剂#1加入剩余的450μL标记试剂#2中，37℃孵育1小时
7) 复染：使用浓度为5mg/mL的DAPI（Sigma D3817），按1:10,000比例稀释于PBS中，室温孵育5分钟
8) 封片：采用ProLong Diamond封片剂（Thermo Fisher Scientific）进行封片
9) 图像采集：使用徕卡Leica SP8倒置激光共聚焦显微镜完成图像拍摄