Cy 5 conjugated anti hrp and hrp or fluorescent coupled secondary antibodies

Male larval body wall muscle preparations were stained as described previously12 (link)41 (link) for CAC1 immunostaining we proceed as described in ref. 31 (link). Primary antibodies used were: rabbit anti-glutamate receptor III (GLURIII) (1:200) (kindly provided by Dr. Tobias Rasse and Dr. David Featherstone), rabbit anti- glutamate receptor IIB (GLURIIB) (1:300, gently provided by Dr. David Featherstone), mouse anti GLURIIA (8B4D2, 1:800, C. Goodman) and mouse anti-Brp (nc82; 1:200, E. Buchner) purchased from Developmental Studies Hybridoma Bank; Cy-5 conjugated anti-HRP and HRP- or fluorescent-coupled secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA) and mouse anti-GFP 3E6 (1:200, Invitrogen, West Grove, PA). After immunocytochemical procedures samples were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Images were captured using a confocal microscope (Olympus FV1000), deconvolved using Huygens Software (Scientific Volume Imaging, Hilversum, The Netherlands) and quantified using ImageJ (U. S. National Institutes of Health). Data was collected in Excel (Microsoft) to be processed. Each data point corresponds to the average quantification of the clusters of at least three boutons from two pictures per larva. The number of data corresponds to the number of larvae imaged.

The immunofluorescence (IF) of the adult brains was performed using the previously described protocols [63 (link)]. Anti-GFP (Invitrogen), Bruchpilot (nc82, DSHB), Futsch (22C10, DSHB), Repo (8D12, DSHB), and Elav (Elav-9F8A9, DSHB) antibodies were employed. The secondary antibodies were purchased from Jackson Laboratories and used 1:200. The larval dissections were performed and the NMJ immunofluorescence of the larvae was performed as in Astorga et al., 2016. The Cy-5-conjugated anti-HRP and HRP- or fluorescent-coupled secondary antibodies (1:200; Jackson ImmunoResearch, West Grove, PA, USA) and mouse Anti-GFP 3E6 (1:200, Invitrogen, Waltham, MA, USA) were employed.