Immunohistochemical Analysis of Larval Neuromuscular Junctions

Wandering third instar larvae of adult flies were randomly selected, dissected in PBS and then fixed in 4% formaldehyde in PBS for approximately 15 min. After blocking with 5% normal goat serum in PBS-T (0.3% Triton X-100) for 1 h, the antibodies with 5% normal goat serum in PBS-T were incubated with the fixed larvae for approximately 1.5 h at room temperature. Larval preparations were mounted with a SlowFade Antifade Kit (Invitrogen). NMJ images were visualized using a laser scanning confocal microscope system (TCS SP5 AOBS/Tandem microscope, Leica-Microscope Systems GmbH, Germany) at Korea Basic Science Institute, Gwangju Center. Leica Application Suite Advanced Fluorescence software was used to analyse images. We performed the analyses of NMJs essentially as described [38 (link)].