Immunocytochemistry.
Immunolabeling of surface-spread spermatocytes and oocytes was performed as described [39,62]. To reach conclusions on the pattern of staining for various proteins, 30 (unless otherwise indicated) well-spread nuclei of particular meiotic stages were first identified under the fluorescent microscope on the basis of SYCP3 or STAG3 staining, then imaged at both appropriate wavelengths to determine the pattern of second proteins with focal patterns such as RAD51 or RPA. Unless otherwise indicated, the panels shown in the figures were the exclusive or predominant patterns seen. The exception for this approach was in the case of staining for MLH1 or MLH3 plus RAD51 (in which case SYCP3 or STAG3 was not available to find chromosome cores). Nuclei in this situation were identified first by MLH1/3 foci clustering, then imaged for both fluorescent wavelengths.
Primary antibodies used in this study were as follows: mouse anti-SCP3 (1:500; Abcam, http://www.abcam.com); rabbit anti-SYCP1 (1:1,000; a gift from C. Heyting) [63]; rabbit anti-REC8 (1:100; a gift from C. Heyting); rabbit anti-RAD51 (1:250, this polyclonal antibody recognizes both RAD51 and DMC1; Oncogene Research Products, http://www.merckbiosciences.co.uk); rabbit anti-γH2AX (1:500; Upstate Biotechnology, http://www.upstate.com/); rabbit anti-STAG3 (1:1,000; a gift from R. Jessberger); rabbit anti-MLH3 (1:400; a gift from P. Cohen); mouse-anti-human MLH1 (1:50; BD Biosciences, http://www.bdbiosciences.com); rabbit-anti-TopBP1 (1:100; a gift from J. Chen) [22]; mouse-anti-ubiquityl-histone H2A (1:200; Upstate Biotechnology); rabbit-anti-TRF2 (1:500; a gift from T. de Lange); and rabbit-anti-BLM (1:50; a gift from R. Freire). All secondary antibodies conjugated with either Alexa Fluor 488 or 594 (Molecular Probes, http://probes.invitrogen.com/) were used at a dilution of 1:1,000. All images were taken with a 100× objective lens under immersion oil.