Fig2c gH2AX and SYCP3 co-staining for Prophase I staging in male meiosis

Raw image dataset for gH2AX and SYCP3 co-staining for Prophase I staging in male meiosis for P21, P28 and adult Wdr62 genetrap testis.
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 spermatocyte spread and immunofluorescence protocol.
Prophase I staging using gH2AX and SYCP3 co-staining:Pacheco,
S. et al. ATR is required to complete
meiotic recombination in mice. Nat Commun
9, 2622, doi:10.1038/s41467-018-04851-z
(2018).

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 or 4% paraformaldehyde 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.