Immunofluorescence experiments for C. elegans were performed as previously described (Zhang et al., 2018 (link)). Images shown in Figure 4—figure supplement 2A were obtained from worms dissected and fixed in the absence of Tween-20 to retain soluble proteins. Primary antibodies were obtained from commercial sources or have been previously described, and were diluted as follows: Rabbit anti-RAD-51 (1:5000, Novus Biologicals, #29480002), Rabbit anti-pHIM-8/ZIMs (1:500, Kim et al., 2015 (link)), Goat anti-SYP-1 (1:300, Harper et al., 2011 (link)), Rabbit anti-SYP-2 (1::500, Colaiácovo et al., 2003 (link)), Chicken anti-HTP-3 (1:500, MacQueen et al., 2005 (link)), Mouse anti-HA (1:400, Thermo Fisher, #26183), Mouse anti-GFP (1:500, Millipore Sigma, #11814460001), Mouse anti-FLAG (1:500, Sigma, #F1804), anti-ALFA-At647N (1:500, Nanotag Biotechnologies, N1502-At647N). Secondary antibodies labeled with Alexa 488, Cy3, or Cy5 were purchased from Jackson ImmunoResearch (WestGrove, PA) and used at 1:500. All images were acquired as z-stacks through 8–12 µm depth at z-intervals of 0.2 µm using a DeltaVision Elite microscope (GE) with a ×100, 1.4 N.A. or ×60, 1.42 N.A. oil-immersion objective. Iterative 3D deconvolution, image projection, and colorization were carried out using the softWoRx package and Adobe Photoshop CC 2021.
Free full text: Click here
