Drosophila Larval Neuromuscular Junction Analysis

Second and third instar wandering Larvae were dissected in ice-cold dissecting saline (HL3) at room temperature and fixed for 5 min in Bouin’s fixative (Sigma Aldrich). Third instar larvae were incubated with primary antibodies for 2 h at room temperature in phosphate buffered saline containing 0.1% Triton X-100. The following primary antibodies were used: mouse anti-Brp (nc82, 1:250), mouse anti-Futsch (22C10, 1:500), mouse anti-Fas II (1D4, 1:60) (all Developmental Studies Hybridoma Bank, Iowa); rabbit anti-GluRIIc (1:3000; Pielage et al., 2011 (link)); rabbit anti-Ank2-XL (1:2000; Koch et al., 2008 (link)); rat anti-Ank2-L (1:40; Enneking et al., 2013 (link)). For second instar larvae primary antibodies were applied overnight at 4°C. Secondary antibodies (Alexa-coupled) were used at 1:1000 for 1 h at room temperature. Conjugated anti-HRP (Jackson Immunoresearch Laboratories) were used at 1:500 together with the secondary antibody for 1 h at room temperature. Larvae were mounted on slides using Prolong Gold antifade reagent.
Images were acquired at room temperature using a Zeiss LSM 700 confocal microscope with a 63 × 1.4 NA oil objective using Zen 2010 software (Zeiss). Calibrated confocal images were used for all quantitative Intensity, diameter and FasII cluster per area measurements and analyzed using FIJI/IMAGEJ and Microsoft Excel.