Dissection and Immunohistochemistry of Drosophila Larval Neuromuscular Junction

Wandering third instars were dissected in Ca²⁺-free saline and fixed in 4% paraformaldehyde for 30 min, except for Ena immunostaining, which required a fixation in a mix of 37% formaldehyde and 100% methanol for 10 min. The following primary antibodies were used for immunohistochemistry: anti-HRP, 1:1000 (123-095-021; Jackson ImmunoResearch, West Grove, PA, USA); FITC-conjugated anti-GFP, 1:1500 (ab6662; Abcam, Cambridge, MA, USA); and anti-Ena 1G6, 1:4 (a gift from F. M. Hoffmann, Madison, WI, USA). Antibodies obtained from the Developmental Studies Hybridoma Bank Iowa City, IA, USA include: anti-α-Spectrin 3A9, 1:30; anti-Cactus 3H12, 1:100; and anti-Dlg 4F3, 1:100. F-actin was visualized using Alexa Fluor 633 phalloidin (1:400; Invitrogen). Secondary antibodies conjugated with fluorophores FITC, Cy3 and AMCA (Jackson ImmunoResearch, West Grove, PA, USA) were used at a 1:200 dilution. RP3 and MN6/7b terminals of muscle 6 and 7 in the abdominal segment A2 of wandering third instar larvae were used for the quantification of all morphological parameters. This analysis was carried out using a Zeiss Axioplan2 microscope and a Hamamatsu ORCA wide-field digital camera as previously described (Loya et al., 2009 (link)). Bouton quantification was performed as previously described (Mosca and Schwarz, 2010 (link)).