Mouse anti brp nc82

Wandering third-instar larvae were dissected, internal organs removed and stretched with insect pins in ice-cold 0-mM Ca²⁺ Haemolymph-like saline (HL3) as previously described58 (link). Larvae were fixed for 10 min with ice-cold 4% paraformaldehyde in PBS solution59 (link) or 3 min in ice-cold methanol for GluRIIA staining, incubated overnight with primary antibodies at 4 °C and incubated with fluorescence-conjugated secondary antibodies at room temperature for 2 h. Primary antibodies were used in the following concentrations: mouse anti-Dlg 1:500 (ref. 55 (link)), rabbit anti-Synaptotagmin 1:2,000 (ref. 60 (link)), rabbit anti-DGluRIIC 1:2,000 (ref. 16 (link)), mouse anti-Brp (Nc82) 1:250, mouse anti-GluRIIA 1:250 (Developmental Studies Hybridoma Bank, USA), rabbit anti-Fur1 1:500 (ref. 37 (link)) and Alexa Fluor 647-conjugated goat anti-horseradish peroxidase (HRP) 1:250 (Jackson ImmunoResearch Laboratories Inc.). Secondary antibodies were used in the following concentrations: Alexa Fluor 488-conjugated goat anti-mouse 1:500, Alexa Fluor 488-conjugated goat anti-rabbit 1:500, Cy3-conjugated goat anti-mouse 1:500 and Cy3-conjugated goat anti-rabbit 1:500 (Amersham Bioscience).

Dissection and antibody labeling was performed as described previously [80 (link)]. Briefly, flies were incubated in 4% PFA + 0.2% Triton-X 100 (Merck, Darmstadt, Germany) for 3.5 hours at 4°C and then washed for 3 × 30 minutes in PBS + 0.2% Triton-X 100 at room temperature prior to brain dissection. Dissected brains were incubated in primary antibodies for 2 to 5 days and in secondary antibodies for 2 days at 4°C. Afterwards, brains were washed for at least 3 × 30 minutes. The following antibodies were used in this study: rabbit anti-GFP (A6455, Thermo Fisher, Waltham, MA) (1:1,000); rabbit anti-Dlg [81 (link)] (1:30,000); rat anti-CD8a (MCD0800, Thermo Fisher, Waltham, MA) (1:500); mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank, Iowa City, Iowa) (1:200); rabbit anti-dsRed (632496, Takara, Kyoto, Japan) (1:500); mouse anti-V5 (960–25, Thermo Fisher, Waltham, MA) (1:500); and Alexa488, 568, and 647 coupled secondary antibodies (Thermo Fisher, Waltham, MA) (1:1,000). Brains were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were captured using a Zeiss LSM 700 laser scanning confocal microscope with a 25× (NA 0.8) or 40× (NA 1.3) oil immersion objective (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were processed using Imaris (Bitplane AG, Imaris.oxinst.com/">www.Imaris.oxinst.com) and Adobe Photoshop software (Adobe; www.adobe.com).

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.