Biot et al. 2024

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MEI4, SYCP3 staining in synchronized mouse spermatocytes WT, Spo11KO, Prdm9KO, Iho1KO, Hormad1KO gH2AX and RPA staining in juvenile mouse spermatocytes of WT and Prdm9Mei4KO

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Spermatocyte spreading and Immunostaining Spreads of spermatocyte nuclei were prepared with the dry down technique, as described 92, and immunostaining was performed as described 87. Staging criteria were as follows: pre-leptotene nuclei had weak SYCP3 nuclear signal and no or very weak γH2AX signal; leptotene nuclei were γH2AX-positive and SYCP1-negative; early/mid zygotene nuclei had less than five or nine fully synapsed chromosomes respectively; late zygotene had nine or more fully synapsed chromosomes; and pachytene cells had all chromosomes fully synapsed, but for the sex chromosomes. The following antibodies were used: guinea-pig anti-SYCP3 (1:500), anti-SYCP1 (1:400) and anti-γH2AX (1:10,000). Microscopy Widefield images were acquired using a Zeiss Axioimager 100X Plan Apochromat 1.4 NA oil objective and a Zeiss CCD Axiocam Mrm 1.4 MP monochrome camera (1388 x 1040 pixels, 6.45µm pixel size). Image analysis For quantification, images underwent deconvolution using Huygens Professional version 22.10 (Scientific Volume Imaging). All image analyses were performed using Fiji/ImageJ 1.53t 98. The “MeiQuant” macro was used for focus counting and intensity measurements 93. Briefly, single nuclei were cropped manually. Foci were detected using the Find Maxima function. On-axis and off-axis foci were distinguished on the basis of their localization within and outside a binary mask, respectively. This region of interest was drawn using an automatic SYCP3 axis protein staining threshold. The same mask was used for measuring focus intensity. Each focus was first defined as on- or off-axis, and then the pixel with maximum intensity was automatically detected using the Find Maxima function. Statistical analyses were performed with GraphPad Prism 9 and the nonparametric Mann-Whitney test to compare the number of foci and focus intensity.

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