Immunohistochemistry of Drosophila Larvae

Wandering third-instar larvae of both sexes from various genotypes were dissected and fixed in Bouin’s fixative for 15 min and processed for immunohistochemistry as previously described (Chen et al., 2012 (link)). Confocal images of all genotypes of larvae belonging to the same experimental group were acquired using the same settings with a Zeiss LSM710 confocal microscope and image editing was done using Adobe Photoshop. Primary antibodies used were FITC-conjugated anti-HRP (1:250, Jackson ImmunoResearch Laboratories), guinea pig anti-Ringer (1:250, Mino et al., 2016 (link)), anti-Ac-Tub (1:1000, T7451, Sigma), anti-Tubulin (1:1000, 2144S, Cell Signaling), and mouse monoclonal anti-Dlg (1:1000, 4F3), anti-Brp (1:250; NC82), anti-Futsch (1:1000; 22C10), and anti-GluR IIA (1:250, 8B4D2) were obtained from Developmental Studies Hybridoma Bank (DSHB), University of Iowa. Secondary antibodies conjugated to Alexa 488 and 568 (Invitrogen-Molecular Probes) were used at 1:400 dilution.