Analyzing H3K27me3 in Drosophila Eye Discs

The GMR-GAL4-D driver (Ahmad and Henikoff, 2001b (link)), UASp-H3.3K27M (Ahmad and Spens, 2019 (link)), and GMR-p21 (Ollmann et al., 2000 (link)) lines were used. All crosses were performed at 25°C. Crawling 3rd instar larvae were selected, and eye discs were dissected and fixed in 4% paraformaldehyde/PBST (PBS with 0.1% triton-X100). Fixed tissues were blocked with 10% goat serum/PBST, and incubated with primary antiserum at 4° overnight, and with fluorescently labeled secondary antibodies (1:200 dilution, Jackson ImmunoResearch). All tissues were stained with 0.5 µg/mL DAPI/PBS, mounted in 80% glycerol on slides, and imaged by epifluorescence on an EVOS FL Auto 2 inverted microscope (Thermo Fisher Scientific) with a 10X or 20X objective. Pseudo-colored images were adjusted and composited in Adobe Photoshop and Adobe Illustrator. H3K27me3 signal in the anterior and posterior portions of eye discs was measured as the mean value in a 100 pixel x 100 pixel box using Photoshop.