Anti dlg1

To analyze the NMJ synapse length on larval muscle 8 and surface area of muscle 8, third instar larvae were dissected in HL3 buffer and fixed in Bouin’s for 3 min. After permeabilization and blocking (10% goat serum), immunostaining was performed with anti-Brp (anti-nc82; Developmental Studies Hybridoma Bank (DSHB), clone name: nc82, 1/100) or anti-Discs large 1 (anti-dlg1; DSHB, clone name: 4F3, 1/200) in larvae that selectively express membrane-tethered GFP in motor neurons (OK371-GAL4, UAS-mCD8::GFP; UAS-mCD8::GFP). Images were taken of muscle 8 in abdominal segment 5 using a Leica SP8 laser scanning confocal microscope with 20× Plan-Apochromat objective (0.8 NA). Maximum intensity projections of z-stacks comprising the entire NMJ were used to measure the synapse length and the surface area of muscle 8.

Wandering L3 larvae were labeled for their gender and dissected, fixed in 3.7% paraformaldehyde for 30 min and co-labeled for bruchpilot (Brp) and discs large 1 (Dlg1). Brp was revealed using the primary antibody nc82 (1:125) (Developmental Studies Hybridoma Bank) applied overnight at 4°C and the secondary Alexa 488-labeled goat-anti-mouse antibody (1:125) (Invitrogen Molecular Probes). Discs large was visualized using primary antibody anti-Dlg1 (1:25) (Developmental Studies Hybridoma Bank) conjugated with the Zenon Alexa Fluor 568 Mouse IgG1 labeling kit (Invitrogen), applied according to the manufacturer’s protocol. For Hrp, Syt and Csp labeling, larvae were blocked for 1.5h on 5% NGS-PBS-T (0.3% Triton X-100 in PBS). Primary antibody anti-Hrp (rabbit, 1:750) (Jackson ImmunoResearch), anti-Syt (rabbit, 1:100) (kindly provided by H.Bellen) or anti-Csp (mouse, 1:25) was applied overnight at 4°C, followed by the Alexa 568- or 488-labeled secondary antibodies (1:500) (Invitrogen Molecular Probes).

Guts were dissected in PBS and transferred into 4% PFA immediately after dissection and staining was performed on an orbital shaker. After 45 min of fixation guts were washed once with PBS for 10 min. Antibodies were diluted in 0.5% PBT + 5% normal goat serum. The incubation with primary antibodies (1:250 anti-Dlg-1 [mouse; Developmental studies Hybridoma Bank (DSHB)]; 1:50 anti-Pros [mouse; DSHB]; 1:2000 anti-pH3 [rabbit; Merck Millipore, 06–570]; 1:50 anti-EcR common Ag10.2 [mouse; DSHB]; 1:500 anti-HA High Affinity 3F10 [rat; Merck, Sigma-Aldrich]; 1:1500 anti-ß-Galactosidase preabsorbed [rabbit; Cappel Research Products]) was performed at 4°C over night. After washing with PBS guts were incubated with secondary antibodies (1:500 Goat anti-MouseAlexa647 [Invitrogen]; 1:500 Goat anti-RatAlexa647 [Invitrogen]; 1:500 Goat anti-RabbitAlexa647 [Invitrogen]) and DAPI (1:1000; 100 µg/ml stock solution in 0.18 M Tris pH 7.4; DAPI No. 18860, Serva, Heidelberg) for at least 3 hr at RT. Guts were washed a last time with PBS prior to mounting in Fluoromount-G Mounting Medium (Electron Microscopy Sciences).