Evos fl auto 2 inverted microscope

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.

We dissected wing imaginal discs from late 3rd instar larvae and fixed them for 20 minutes in 4% formaldehyde/PBST (PBS with 0.1% triton-X100).
For quantitative detection of proteins in wing discs, tissues from GFP-positive controls and mutant animals were dissected and incubated with antibodies in the same well, Fixed issues were blocked with 10% goat serum/PBST, and incubated with anti-Vg (1:100 dilution, a gift from S Carroll, University of Wisconsin-Madison) and anti-Wg (1:200 dilution, clone 4D4, Developmental Studies Hybridoma Bank) 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 objective.

Testes from third-instar male larvae were prepared as described [Bonaccorsi et al, 2000 (link)]. Briefly, one testis was dissected in a drop of PBS on a Histobond glass slide (VWR 16004–406), squashed gently with a RainX (ITW Global Brands) coated coverslip, then flash-frozen in liquid nitrogen. After popping off the coverslip, the sample was fixed with 4% formaldehyde/PBST for 5 minutes, and incubated with 0.3% sodium deoxycholate/PBST twice for 20 minutes each. Samples were incubated with primary antiserum in PBST supplemented with 0.1% bovine serum albumin (BSA) at 4° overnight, and finally with fluorescently-labeled secondary antibodies (1:200 dilution, Jackson ImmunoResearch). Slides were stained with 0.5 μg/mL DAPI/PBS, mounted in 80% glycerol, and imaged by epifluorescence on an EVOS FL Auto 2 inverted microscope (Thermo Fisher Scientific) with a 40X objective. Dissection and immunostaining was typically repeated at least 10 times to confirm results. We also imaged stained spermatocytes from adult testes, but imaging of larval spermatocytes was typically cleaner with less background. Pseudo-colored images were adjusted and composited in Adobe Photoshop and Adobe Illustrator.