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.
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