Dabco mowiol 4 88

Wing imaginal disks dissected from third instar Drosophila larvae (7 d after egg laying) were fixed for 25 min with 4% paraformaldehyde in PBS containing 0.1% Triton X (PBS-T) at room temperature. Fixed tissues were washed three times with PBS-T. Primary antibodies were diluted in blocking buffer (PBS-T with 0.3% BSA), and tissues were stained overnight at 4°C. The following primary antibodies were used: rabbit anti-mRFP (1:500, Cat# PM005, RRID:AB_591279; MBL International), goat anti-GFP (1:500, Cat# ab6673, RRID:AB_305643; Abcam), and rabbit-anti HA (1:1,000, Cat# ab9110, RRID:AB_307019; Abcam). After washing, the samples were incubated with the corresponding Alexa Fluor 488 (1:2,000, Cat# A-11034, RRID:AB_2576217; Thermo Fisher Scientific)–, Cy2 (1:2,000, Cat# 705-225-147, RRID:AB_2307341; Jackson ImmunoResearch Labs)-, or Cy3 (1:2,000, Cat# 711-165-152, RRID:AB_2307443; Jackson ImmunoResearch Labs)-conjugated secondary antibodies overnight at 4°C, and washed and counterstained with DAPI (1:1,000 dilution of 5 mg/ml stock, Cat# 6335.1; Carl Roth GmbH) to visualize nuclei. Tissues were mounted on glass slides in DABCO–Mowiol 4-88 (Cat# D2522 and Cat# 81381; Sigma-Aldrich).

EADs and wing imaginal discs dissected from third instar Drosophila larvae (7 days AEL) were fixed for 25 min with 4% paraformaldehyde in PBS containing 0.1% Triton X (PBS-T) at room temperature. Adult female intestines were fixed with 4% paraformaldehyde in PBS for 48 h at 4°C. Fixed tissues were washed three times with PBS-T, intestines in PBS only. Primary antibodies were diluted in blocking buffer (PBS-T with 0.3% BSA) and tissues were stained overnight at 4°C. The following primary antibodies were used: rabbit anti-Drosophila cleaved Death caspase 1 (Dcp-1, 1:500, Cell Signaling Technology Cat #9578, RRID:AB_2721060), rat-anti Ecd N-term (1:500, 22 (link)), mouse-anti Armadillo (Arm, 1:20, Developmental Studies Hybridoma Bank #N2 7A1; RRID:AB_528089), mouse-anti disc large (Dlg1, 1:100, Developmental Studies Hybridoma Bank #4F3; RRID:AB_528203). After washing, the samples were incubated with the corresponding Cy3- or Cy5-conjugated secondary antibodies (1:1000, Jackson ImmunoResearch Labs Cat# 711-175-152, RRID:AB_2340607, Cat# 712-165-150, RRID:AB_2340666, Cat# 715-165-151, RRID:AB_2315777) for 2 h at room temperature and counterstained with DAPI (1:1000 dilution of 5 mg/ml stock, Carl Roth GmbH Cat# 6335.1) to visualize nuclei. Tissues were mounted on glass slides in Dabco-Mowiol 4–88 (Sigma-Aldrich Cat# D2522 and Cat# 81381) and imaged within 72 h.