Alexa fluor 488 goat

Meiocytes from A. suecica (JC2) were embedded in acrylamide to preserve their three‐dimensional structure and then used for the immunolocalization studies, as previously described (Nibau et al., 2020 (link); Phillips et al., 2010 (link)). Briefly, young buds (<1 mm) were harvested and fixed in 2% (w/v) paraformaldehyde, washed, macerated with a brass rod in 1% (v/v) Lipsol in buffer A and embedded in acrylamide. Embedded meiocytes were blocked and incubated with α‐ASY1 (rat, 1:500; Armstrong et al., 2002 (link)), α‐ZYP1 (guinea pig, 1:500; Higgins et al., 2005 (link)) and α‐MLH1 (1:250; Chelysheva et al., 2010 (link)) antibody solution for 24–36 h. After washing, embedded meiocytes were incubated overnight with secondary antibodies (Alexa Flour 568 goat anti‐rat, A11077, Alexa Fluor 488 goat anti‐rabbit, A32731, and Alexa Fluor 633 goat anti‐guinea pig, A21105; Invitrogen, now ThermoFisher Scientific), used at 1:500 dilution. Images were acquired using a Leica TCS SP8 confocal microscope (https://www.leica‐microsystems.com) with maximum projection of Z‐stacks and deconvolved using the built‐in lightning software. Image analysis was carried out in imaris 7.3 (Oxford Instruments, https://imaris.oxinst.com). Diakinesis and diplotene cells were identified for image acquisition based on the presence of ASY1 signal and MLH1 foci and the absence of ZYP1 signal.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X and blocked with 5% BSA at room temperature. Cells were incubated with anti-Phospho Histone γH2AX primary antibody (1:500) and then with secondary antibody (1:1000) anti-rabbit immunoglobulin G (Alexa Fluor™ 488 goat, A11008, Invitrogen, Carlsbad, CA, USA). Images were captured with the fluorescence microscope Olympus IX51 (400× magnification) with digital camera Olympus XM10, using the CellSens software. Quantification was performed in Image J, by measuring signal intensity and normalizing to the number of cells assessed.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X and blocked with 5% bovine serum albumin at room temperature. Fixed cells were incubated firstly with primary antibody (Table S3) and then with secondary antibody anti-rabbit immunoglobulin G (Alexa FluorTM 488 goat, A11008, Invitrogen, Carlsbad, CA, USA). DNA staining was performed with 4′,6-diamidino-2-phenylindole (Boster Biological Technology, Pleasanton, CA, USA). Pictures were taken in a fluorescence microscope Olympus IX51 (400× magnification) with digital camera Olympus XM10, using CellSens software.