Synapsed homologs of meiotic mouse chromosomes visualized by TIRFM

Mouse testes were removed from euthanized animals, followed by decapsulation and maceration in high-glucose MEM medium. Suspension was mixed thoroughly and left to settle; the supernatant was then collected and centrifuged at 7200 rpm for 1 min. The pellet was then resuspended in a 0.5 M sucrose solution and added to PFA-treated (1% in 0.015% Triton X-100) coverslides, which were incubated for at room temperature for 2 hours in a humidified environment. After incubation, slides were rinsed twice with a wetting agent solution (Kodak, 1464510) in water and allowed to air dry. SYCP3 was labeled with primary SCP-3 (D-1) antibody (Santa Cruz Biotechnology, SC-74569) and a secondary anti-mouse antibody fused with Alexa-568 (Thermo, A11004). Surface chromosome spreads were imaged in an Elyra 7 microscope (Zeiss), with a 60x 1.4 NA oil immersion objective and an a 1.4x magnification lens. Image reconstruction was performed in ZEN Black with parameters set to default. Experimental procedures were approved by the “Ministero della Salute" of Italy, authorization n.701/2018-PR.