Fig4c Centrin-SYCP3 and CEP63 co-staining on adult spermatocyte spreads

Raw image dataset for Centrin-SYCP3 and CEP63 co-staining on Wdr62 genetrap adult spermatocyte spreads.
Please see Becherel, O. J. et al. Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing. PLoS Genet 9, e1003435, doi:10.1371/journal.pgen.1003435 (2013) for protocol on making spermatocyte spreads.
Briefly, testes were decapsulated, chopped and resuspended in DMEM (Gibco) before passing through 100μm cell strainer. Cells were pelleted by centrifuging at 500rpm at room temperature for 5mins and then resuspended in 0.1M sucrose. 10μL of cell suspension was spread onto glass slides pre-wetted in 100% methanol and fixed for 2 hrs. The slides were subsequently washed with PBS and air-dried in the presence of Kodak Photo-Flo 200 (Kodak, 1 in 250 dilution). Slides were stored at -80°C. For immunostaining, slides were rehydrated in PBS and blocked in blocking buffer (20% FBS, 0.2% Triton X in PBS) for 1 hr before incubating with primary antibody at 4°C overnight. The next day, slides were incubated with secondary antibody and counterstained with DAPI before mounting in Prolong Diamond (Invitrogen). The primary and secondary antibodies used are listed in Supplementary Table 1.

本数据集为针对Wdr62基因陷阱（genetrap）成年精母细胞铺片的中心蛋白（Centrin）-联会复合体蛋白3（SYCP3）与CEP63共染色原始图像数据集。

如需获取精母细胞铺片的实验流程，请参阅Becherel O.J.等人2013年发表于*PLoS Genet*（《公共科学图书馆·遗传学》）的研究：《Senataxin与DNA损伤应答蛋白在减数分裂重组及基因沉默中发挥关键作用》，刊载于第9卷，文章编号e1003435，DOI:10.1371/journal.pgen.1003435。

简要实验步骤如下：先将睾丸被膜剥离、切碎后重悬于DMEM（Gibco）培养基中，经100μm细胞筛过滤；室温下以500rpm离心5分钟收集细胞沉淀，并重悬于0.1M蔗糖溶液中；取10μL细胞悬液滴加到预先以100%甲醇润湿的载玻片上，固定2小时；随后用磷酸盐缓冲液（PBS）洗涤载玻片，在Kodak Photo-Flo 200（柯达，1:250稀释液）中自然晾干；制备完成的载玻片保存于-80℃冰箱。

免疫染色实验时，先将载玻片置于PBS中复水，再用封闭缓冲液（20%胎牛血清（FBS）、0.2% Triton X溶于PBS）封闭1小时，随后与一抗于4℃孵育过夜；次日，将载玻片与二抗孵育，并用4',6-二脒基-2-苯基吲哚（DAPI）复染，最后使用Prolong Diamond（Invitrogen）进行封片。本实验所用一抗及二抗信息详见补充表1。