Mouse anti γh2ax

Meiotic cells were prepared as previously described by Massip et al. (2010) [25 (link)]. The synaptonemal complex proteins 3 (SCP3) and 1 (SCP1), MutL homolog 1 protein (MLH1) and centromeres were detected using the following primary antibodies: rabbit anti-SCP3 (1:1000; ABCAM, Cambridge, UK), rabbit anti-SCP1 (2:1000; ABCAM, Cambridge, UK), mouse anti-MLH1 (2:100; Becton Dickinson, Franklin Lakes, NJ), Human anti-kinetochore (1:100; Antibodies Incorporated, Davis), respectively, and prepared in a solution of PBT (PBS +0.16% BSA +0.1% Tween). The secondary antibodies consisted of Alexa 594 conjugated donkey anti-rabbit (1:100, Molecular Probes), Alexa 488 conjugated goat anti-mouse (1:100, Molecular Probes, Eugene, OR, USA), and AMCA conjugated donkey anti-human (1:100, Jackson Immunoresearch, West Grove, PA, USA). The γH2AX protein was detected by carrying out a complementary experiment without MLH1 antibody but with mouse anti-γH2AX (ABCAM, Cambridge, UK) and Alexa 488 conjugated goat anti-mouse (1:100, Molecular Probes, Eugene, OR, USA) antibodies. γH2AX is considered as a marker of unsynapsed chromatin (transcriptionally silenced chromosome regions—Turner et al. (2005) [26 (link)])

Meiotic cells were prepared as described by Pinton et al. (2008 (link)) with some modifications. Detection of the synaptonemal complex proteins 3 (SCP3) and 1 (SCP1) and centromeres was carried out before immunostaining the γH2AX protein.
The meiotic proteins were immunolocalized using antibodies at 1:100 dilution in PBT (1× phosphate-buffered saline (PBS), 0.15 % bovine serum albumin (BSA), and 0.1 % Tween 20) as follows. First, the SCP1 and centromeres were detected using the following primary antibodies: rabbit anti-SCP1 (Abcan, Cambridge, UK) and human anti-centromere (Antibodies Incorporated, Davis, CA, USA). Secondary antibodies consisted of DyLight 488 conjugated goat anti-rabbit (KPL, Gaithersburg, MD, USA) and 1-amino-4-methylcoumarin-3-acetic acid (AMCA) conjugated donkey anti-human (Jackson ImmunoResearch Laboratories, Grove, PA, USA). Secondly, SCP3 was detected using rabbit anti-SCP3 (Abcam, Cambridge, UK) and then revealed with secondary antibody Alexa 594 conjugated donkey anti-rabbit (Molecular Probes, Eugene, OR, USA). Spermatocytes were captured using a Zeiss Imager Z2 microscope with CytoVision imaging system (Leica Microsystemes, Nanterre, France). Finally, the γH2AX protein was detected using mouse anti-γH2AX (Abcam, Cambridge, UK) and Alexa 488 conjugated goat anti-mouse (Molecular Probes, Eugene, OR, USA) antibodies.

Cells were grown in chamber slides, treated, fixed, immunostained, and analyzed as previously described [60 (link)]. Cells with more than 10 foci were determined as positive. The primary antibodies used were mouse anti-γH2AX (Abcam) at a dilution of 1:2,000, rabbit anti-53BP1 (Cell Signaling) at a dilution of 1:500, and rabbit anti-RPA70 (Cell Signaling) at a dilution of 1:500. Secondary anti-mouse Alexa 555 and anti-rabbit Alexa 488 were from Invitrogen and were used at a dilution 1:1000.