Metathoracic legs were cut at the coxal segment and fixed in 4% formaldehyde for 45 min prior to dissection. After washing 3× in PBS, a cuticle patch along the femur was removed exposing underlying tissue and samples were post-fixed for 15 min in 4% formaldehyde. Antibody staining protocols followed standard methods and samples were mounted in slow-fade diamond mount (Molecular Probes) (Patel, 1994 (link)). Antibodies and stains used were anti-horse-radish peroxidase (#115-035-166, Jackson ImmunoResearch, 1:550), anti-dlg (4F3, Developmental Studies Hybridoma Bank, 1:200), phalloidin-633 (Molecular Probes, 1:1000), mouse anti-FK2 monoclonal (#BML-PW0150-0025, Enzo Life Sciences, 1:200); mouse anti-Bruchpilot monoclonal (nc82, Developmental Studies Hybridoma Bank, 1:20), mouse anti-ATP5a (#ab14748, Abcam, 1:200). Imaging was performed by confocal microscopy (Olympus FV1000) capturing z-stacks for all samples. Image processing occurred in ImageJ (FIJI) and consisted of cropping images for areas of interest and altering brightness/contrast as needed for publication. Where necessary image size was increased using linear transformation. Image stitching was performed in ImageJ as needed (Preibisch et al., 2009 (link)).
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