Testicular Nuclei Preparation and Immunostaining

Surface-spread testicular nuclei were prepared using the hypotonic treatment protocol described previously [51 (link)] with a few modifications [36 (link),38 (link)]. The following antibodies were used: rabbit polyclonal anti-RAD51 (Santa Cruz, sc-8349), anti-DMC1 (Santa Cruz, sc-22768), and anti-SYCP1 (Abcam, ab15087); mouse monoclonal anti-γH2AFX (Upstate, #05–636) and anti-SYCP3 (Santa Cruz, sc-74569); goat anti-Rabbit IgG Alexa Fluor 488 (Molecular Probes, A-11034); and goat anti-Mouse IgG Alexa Fluor 568 (Molecular Probes, A-11031) and IgG Alexa Fluor 647 (Molecular Probes, A-21236). The images were acquired using a Nikon E400 microscope with a DS-QiMc mono-chrome CCD camera (Nikon) and processed in the NIS-Elements program (Nikon). The spread nuclei were staged based on axis development and synaptonemal complex formation.

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Baker C.L., Petkova P., Walker M., Flachs P., Mihola O., Trachtulec Z., Petkov P.M, & Paigen K. (2015). Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots. PLoS Genetics, 11(9), e1005512.

Publication 2015

anti-RAD51 anti-DMC1 anti-SYCP1 goat anti-Rabbit IgG Alexa Fluor 488 IgG Alexa Fluor 647 DS-QiMc mono-chrome CCD camera NIS-Elements program

Alexa 568 Alexa fluor 488 Alexa fluor 647 Anti igg Antibodies Axis Dmc1 Goat Microscope Molecular probes Mouse Nuclei Rabbit Synaptonemal complex Testicular Treatment protocol

Corresponding Organization : Czech Academy of Sciences

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Variable analysis

independent variables

Hypotonic treatment protocol with modifications

dependent variables

Localization and expression of RAD51, DMC1, SYCP1, γH2AFX, and SYCP3 proteins

control variables

Rabbit polyclonal anti-RAD51, anti-DMC1, and anti-SYCP1 antibodies
Mouse monoclonal anti-γH2AFX and anti-SYCP3 antibodies
Goat anti-Rabbit IgG Alexa Fluor 488, goat anti-Mouse IgG Alexa Fluor 568 and Alexa Fluor 647 secondary antibodies
Nikon E400 microscope with DS-QiMc monochrome CCD camera and NIS-Elements software

positive controls

None specified

negative controls

None specified

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