Meiotic Chromosome Synapsis Assay

Surface spreading of meiotic nuclei was performed as described by Grubb et al., 2015 (link). During spheroplasting, 20 μl of dithiothreitol (DTT) was used instead of the published 40 μl. Fixation was achieved with 4% PFA/sucrose solution. Spreads from Figure 3J,M were stained with anti-Zip1 guinea pig polyclonal antibody (a gift from Scott Keeney, 1:500 dilution), and then with goat anti-guinea pig polyclonal secondary antibody (Alexa Fluor 555; ThermoFisher; A-21435, 1:200 dilution). Spreads from Figure 3—figure supplement 2J were stained with anti Zip1 goat polyclonal antibody (Santa Cruz Biotechnology; y-N16, 1:50 dilution), and then with donkey anti-goat polyclonal secondary antibody (Alexa Fluor 555; ThermoFisher; A-21432, 1:1000 dilution). Slides were mounted in antifade with added DAPI (Prolong Gold, Invitrogen; P36930) and images were captured using a Zeiss AxioPlan II microscope, Hamamatsu ORCA-ER CCD camera and Volocity software. Synapsis was quantified by characterizing four classes of Zip1 staining pattern: no Zip1, foci only, a mixture of foci and lines, and lines only (as previously described by Chen et al., 2015 (link)).