Immunostaining Analysis of Meiotic Structures

Meiotic nuclei were surface spread on glass slides and imaged as described in [24 (link)]. The following primary antibodies were used: affinity purified rabbit anti-Zip1 (1:100, raised at YenZym Antibodies, LLC, against a C terminal fragment of Zip1, as described in [43 (link)], mouse anti-cMYC (1:200, clone 9E10, Abcam). Mouse anti-Gmc2 antibodies were raised against purified Gmc2 protein, and guinea pig anti-Gmc2_Ecm11 antibodies were raised against a co-purified protein complex (ProSci Inc.). These antibodies were used at 1:800. Chicken anti-HA (1:100, Abcam), and rabbit anti-Red1 (1:100, a kind gift from G.S. Roeder, [59 (link)]) were also used. Secondary antibodies conjugated with Alexa Fluor dyes were purchased from Jackson ImmunoResearch and used at 1:200 dilution. Microscopy and image processing were performed using a Deltavision RT imaging system (General Electric) adapted to an Olympus (IX71) microscope. Measurements of Zip1, Ecm11, and SC linear structures in Fig 4 and S2 Fig (raw data in S5 Table) were measured manually by K.V.M. (“ImageK”), using the measurement tool in the SoftWorx program associated with the Deltavision RT system. Structured illumination microscopy was carried out using Applied Precision’s OMX Blaze Structured Illumination Microscope system at The Rockefeller University’s Bio-Imaging Resource Center.