Immunolabeling of Meiotic Chromosome Spreads

Spermatocytes for surface spreading were prepared from testes of ~2-month-old mice using established methods^{80 (link)}. The following primary antibodies were used in dilution buffer (0.2% BSA, 0.2% fish gelatin, 0.05% Triton X-100, 1× PBS), with incubation overnight at 4°C: goat anti-SYCP3 (Santa Cruz Biotechnology Cat# sc-20845; 1:200), rabbit anti-RAD51 (Calbiochem Cat# PC130; 1:200), rabbit anti-DMC1 (Santa Cruz Biotechnology Cat# sc-22768; 1:200). This was followed by incubation with the following secondary antibodies at 1:500 dilution for 1 h at 37°C: 488 donkey anti-rabbit (Life Technologies Cat#A21206), donkey 594 anti-goat (Invitrogen Cat# A11058). Coverslips were mounted with ProLong Gold antifade reagent with DAPI. Immunolabeled chromosome spread nuclei were imaged on a Deltavision microscope using ×100 oil-immersion objective. Only foci colocalizing with the chromosome axis were counted.

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Prakash R., Sandoval T., Morati F., Zagelbaum J.A., Lim P.X., White T., Taylor B., Wang R., Desclos E.C., Sullivan M.R., Rein H.L., Bernstein K.A., Krawczyk P.M., Gautier J., Modesti M., Vanoli F, & Jasin M. (2021). Distinct pathways of homologous recombination controlled by the SWS1–SWSAP1–SPIDR complex. Nature Communications, 12, 4255.

Publication 2021

goat anti-SYCP3 rabbit anti-DMC1

Antibodies Axis Buffer Chromosome Dapi Dilution Dmc1 Donkey Fish Gelatin Goat Gold Immersion Mice Microscope Nuclei Rabbit Spermatocytes Testes Triton x 100

Corresponding Organization : Memorial Sloan Kettering Cancer Center

Other organizations : Inserm, Aix-Marseille Université, Centre National de la Recherche Scientifique, Columbia University, Amsterdam University Medical Centers, UPMC Hillman Cancer Center

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Variable analysis

independent variables

Age of mice (approximately 2 months old)

dependent variables

Localization and colocalization of SYCP3, RAD51, and DMC1 proteins on chromosome spreads

control variables

Preparation of spermatocytes for surface spreading using established methods
Dilution buffer composition (0.2% BSA, 0.2% fish gelatin, 0.05% Triton X-100, 1× PBS)
Incubation of primary antibodies overnight at 4°C
Incubation of secondary antibodies for 1 h at 37°C
Mounting of coverslips with ProLong Gold antifade reagent with DAPI
Imaging of immunolabeled chromosome spread nuclei on a Deltavision microscope using ×100 oil-immersion objective
Counting of only foci colocalizing with the chromosome axis

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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