Deltavision ix70 system

Images were captured on the Deltavision IX70 system (Applied Precision; Olympus) with a 100× objective lens (NA 1.40) and a camera (CoolSNAP HQ2; Photometrics) using softWoRx software at RT. 7 consecutive z-slices with an interval of 0.2 µm were acquired, then processed by deconvolution using the constrained iterative deconvolution algorithm within softWoRx. The deconvolved z-slices were projected using a maximum-intensity algorithm to form the final processed image. Meiotic chromosomes were surface spread as described previously (Dresser and Giroux, 1988 (link)), except that glass slides were not precoated with plastic. In brief, meiotic cells from appropriate time points were digested with zymolyase in the presence of 1 M sorbitol to form spheroplasts. Cells were gently washed once, and chromosome spreads were prepared by resuspending spheroplasts in buffered solution without sorbitol (22 mM MES) and 3% PFA. Cell suspension was spotted onto clean glass slides and left for 20 min for spread chromosomes to settle. The slides with chromosome spreads were washed with PBS once and subjected to standard immunostaining protocols. Primary antibodies used were Zip1 and Red1 (rabbit/mouse, 400-fold dilution, S. Roeder (Yale University, New Haven, CT); Sym et al., 1993 (link); Smith and Roeder, 1997 (link)). Secondary antibodies were Alexa Fluor 488 or 594 (Invitrogen).

For cytology, 1 × 10⁸ cells were harvested at the indicated time point and yeast chromosome spreads were prepared as described in Grubb et al. (2015) (link). Primary antibodies used were mouse monoclonal 9E11 anti-myc antibody (dilution 1:200), rabbit polyclonal anti-Zip1 antibody (dilution 1:100; Santa Cruz Biotechnology sc-33733), rabbit monoclonal anti-Red1 antibody (#16441; dilution 1:200; a gift from N. Hollingsworth), and rabbit anti-Gmc2 antibody (dilution 1:1600; a gift from Amy MacQueen). The secondary antibodies were Alexa488-conjugated goat antirabbit (dilution 1:200; Thermo Fischer Scientific A-11008) and Alexa568-conjugated goat antimouse (dilution 1:200; Thermo Fischer Scientific A-11004). Chromosomal DNA was stained by 4,6-diamidino-2-phenylindole (DAPI). Fluorescence images were visualized and acquired using the Deltavision IX70 system (Applied Precision), 100× objective, and softWoRx imaging software. Images were processed by deconvolution using the constrained iterative deconvolution algorithm within softWoRx. Image analysis and signal quantification were performed using the Fiji software and R scripts. Fluorescence intensity was measured as the average sum of pixel density of Zip1 stretches per nucleus.