Cfi apochromat tirf oil immersion lenses

Slides were incubated in PBT (0.15% BSA, 0.1% Tween 20 in PBS) for 60 min prior to overnight incubation at room temperature with the antibodies listed in supplementary material Table S1. Thereafter, slides were washed 3 times for 5 min each in PBS with 0.1% Tween 20, incubated with secondary antibodies (Invitrogen or Jackson ImmunoResearch) at 1:500 for 60 min in PBT, washed in PBS plus 0.1% Tween 20, and mounted in Vectashield with DAPI. Images of germ cells were acquired with an ECLIPSE Ti-E microscope (Nikon) and Zyla 5.5 sCMOS camera (Andor Technology), with 20×, 60×, and 100× CFI Apochromat TIRF oil immersion lenses (Nikon), numerical aperture 1.40; image acquisition was performed using NIS-Elements Basic Research software (Nikon). Images were taken at RT (∼22°C). Phylum, Volocity 3D Image Analysis (PerkinElmer), NIS-Elements Basic Research, and NIS-Elements Viewer (Nikon) were used for image analysis. Photoshop and Illustrator (Adobe) were used for composing figures. Particular stages of primary spermatocytes were determined by staining for H2AX, SYCP3 and/or H1T. For data analysis, the matched substage of meiosis was analyzed in controls and mutants. All data were confirmed with at least two or three independent mice. Total numbers of analyzed nuclei in at least two independent experiments are shown in each panel.