Immunostaining of Meiotic Cell Spreads

Meiotic cell spreads were prepared using the method of Peters et al. (1997) (link) with minor modifications as described (Dumont et al. 2015 (link)). Spermatocyte cell spreads were immunostained according to a protocol adapted from Anderson et al. (1999) (link) and Koehler et al. (2002) (link) as previously described (Dumont et al. 2015 (link)). The primary antibodies used were as follows: mouse anti-MLH1 (1:100 dilution; BD Biosciences), goat anti-SYCP3 (1:100 dilution; Santa Cruz Biotechnology), rabbit anti-phospho-histone H2A.XpSer139 (1:1000 dilution; Thermo Scientific), human anti-centromere (1:100 dilution; Antibodies Incorporated), and rabbit anti-SYCP1 (1:100 dilution; Abcam). The following secondary antibodies were used at 1:200 concentration: donkey anti-goat Rhodamine Red-X, donkey anti-rabbit Alexa Fluor 488, donkey anti-human Aminomethylcoumarin Acetate, and donkey anti-mouse Alexa Fluor 488 (Jackson ImmunoResearch).