Mouse anti bruchpilot nc82

Dissected brains were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed tissue was blocked with PBS containing 2% Triton X-100 and 10% normal goat serum (NGS) for 30 min and then incubated for 1 day each (with washing in between) at room temperature in PBS containing 1% Triton X-100, 0.25% NGS, and a primary antibody or secondary antibody solution. The brain was mounted in 20 μl Vectashield medium. We used the following primary antibodies at the indicated dilutions: 1:50 mouse anti-bruchpilot (nc82) (Developmental Studies Hybridoma Bank, Iowa), 1:1000 rabbit anti-5HT (Sigma, S5545). Secondary antibodies from Invitrogen were used at dilutions of 1:250, which were Alexa Fluor 633 goat anti-mouse lgG (Life Technologies, A21050), Alexa Fluor 568 goat anti-mouse lgG (Life Technologies, A11004), Alexa Fluor 633 goat anti-rabbit (Invitrogen, A21071). Confocal images were acquired with Zeiss LMS710 confocal laser scanning microscope under 40x or 63x magnification.

Third-instar larvae were dissected and fixed in 4% paraformaldehyde (except Bouin’s fixative was used for GluRIIA staining). Fixed samples were washed with 0.1% triton X-100 in PBS (PBST) then blocked with 5% normal goat serum (NGS) in PBST. Primary antibodies used to label dissected larvae were diluted in blocking solution and used as following: rabbit anti-p-Synj, 1:2000^{30 (link)}; guinea-pig anti-Endo(GP69), 1:200^{40 (link)}, guinea-pig anti-Dap160, 1:1000^{33 (link)}; rabbit anti-synaptotagmin, 1:1000^{69 (link)}, rabbit anti-Mnb, 1:400; rabbit anti-Synj-1, 1:200; mouse anti-dynamin, 1:200 (BD Transduction Laboratories); Cy3 conjugated anti-HRP, 1:100 (Jackson ImmunoResearch); mouse anti-bruchpilot (NC82), 1:10; mouse anti-GluRIIA, 1:50 and mouse anti-Dlg (4F3), 1:500 (Developmental Studies Hybridoma Bank). Secondary antibodies used were Alexa-488, 555 or 405 conjugated, 1:250 (Invitrogen).