Alexa fluor 488 coupled goat anti rabbit

C. elegans dauer larvae and post-dauer adult gonads were dissected, fixed, and stained as described elsewhere [45 (link)]. Extruded gonads were incubated with rabbit anti-H3K4me3 (1:500 dilution, Abcam, ab8580) or rabbit anti-H3K9me3 (1:500 dilution, Cell Signalling Technology, 9754S). Secondary antibodies were Alexa-Fluor-488–coupled goat anti-rabbit (1:500; Life Technologies, Carlsbad, CA, USA). Gonads were counterstained with DAPI (0.1 μg/mL dilution, Roche Diagnostics, 10236276001). Microscopy was performed as described elsewhere [10 (link),46 (link)]. Ratios for the fluorescence intensity across the germ line were determined using ImageJ. The ratio was calculated by dividing the immunofluorescence signal of either anti-H3K4me3 or anti-H3K9me3 by the fluorescence signal of DAPI per nucleus.

For extruded dauer gonad staining, gonads were dissected, fixed, and stained as described elsewhere [64 (link)]. The following primary antibodies were used: rabbit polyclonal anti-H3K4me3 (1:500; Abcam, Cambridge, UK), anti-H3K9me3 (1:500; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-HIM-3 (gift from Zetka lab, 1:200). Secondary antibodies were Alexa-Fluor-488–coupled goat anti-rabbit (1:500; Life Technologies, Carlsbad, CA, USA). Microscopy was performed as described in [65 (link)]. Ratios for the fluorescence intensity across the germ line were determined using Image J.