Chromosome spreads preparation and immunostaining

Overall, 5 × 10⁷ diploid cells (5 ml O.D. 1) were collected at different time points during pat1-114 synchronous meiosis. Spreads were prepared essentially as described in (Loidl and Lorenz, 2009) and stored at −20 °C until use. Immunostaining was performed as described in (Loidl and Lorenz, 2009). Primary and secondary antibodies were incubated at RT, overnight and 4 h, respectively. Antibodies used were: rabbit IgG fraction anti-GFP (1:800; A11122 Molecular Probes), monoclonal anti-Rad51 (Clone 51RAD01/3C10) (1:50, Invitrogen), anti-mouse Alexa 488 (1:1000; A11001 Molecular Probes) and anti-rabbit Alexa 568 (1:1000; A11011 Molecular Probes). Specificity of the primary antibodies was tested by staining sfr1⁺ rec12 deleted cells (Appendix Fig. S7). Images were acquired under Nikon Ti2-E spinning disk microscope equipped with a confocal Dragonfly module (ANDOR), a 100×/1.45 Oil Plan APO lens, a sCMOS Sona 4.2B-11 camera (ANDOR) and Fusion 2.2 software (ANDOR). In all, ×1.5 magnification and deconvolution were applied during acquisition. 16 Z sections were collected to cover 3 µm of total thickness (0.2 µm step size). Rad51 and EGFP-Sfr1 foci analysis was performed with Fiji software (Schindelin et al, 2012) using the sum Z projection. Mean intensity and number of foci were measured using Trackmate plug-in with an object diameter of 0.3 µm. A constant threshold among samples was set to count foci with significant intensity, but a weaker signal was always observed and uniformly distributed along chromatin. Colocalization percentage was calculated by counting overlapping Rad51 and EGFP-Sfr1 foci. For further colocalization analysis, Pearson’s coefficient was determined using JACoP plug-in, and Costes randomization was applied in all cases to discard spurious colocalization (Costes P value = 100 in all cases).