Fig4a Centrin-SYCP3 and PCNT co-staining on adult spermatocyte spread

Raw image dataset for Centrin-SYCP3 and PCNT co-staining on Wdr62 genetrap adult spermatocyte spread.
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.