Mouse anti brp antibody

For analysis of neuromuscular junction (NMJ) phenotypes in Drosophila, wandering third instar larvae were dissected as previously described (Smith and Taylor, 2011 (link)). Dissected larvae were fixed in 3.7% formaldehyde in PBS for 20 min, washed in PBT (PBS with 0.4% Triton X-100) and blocked with 10% normal goat serum for 1 hr at room temperature. The larvae were then incubated overnight at 4°C with mouse anti-Brp antibody (1:50; Developmental Studies Hybridoma Bank, nc82; RRID:AB_2314865) to label the active zones and Cy3 goat anti-HRP (1:200; Jackson ImmunoResearch, 123-165-021; RRID:AB_2338959) to label the NMJ membrane and axons. Next, samples were washed in PBT and incubated with Alexa Fluor 488 goat anti-mouse (1:200; Molecular Probes, A11001; RRID:AB_2534069) in PBT + 10% normal goat serum at room temperature for 2 hr. Multiple confocal stacks were acquired using a point scanning confocal microscope (Leica TCS SPE) and then processed using the DeadEasy Synapse ImageJ plugin (Sutcliffe et al., 2013 (link)), which provides total voxel occupancy by Brp signal at each nerve terminal. Brp signal was quantified at muscle 6/7 NMJs from abdominal segment A3.

The primary antibodies used in the immunostaining experiments were as follows: mouse anti-GFP antibody (1:500, Roche, no. 11814460001), chicken anti-mCherry antibody (1:300, Novus Biologicals, no. NBP2-25158) and mouse anti-BRP antibody (1:100, Developmental Studies Hybridoma Bank, no. nc82). The secondary antibodies for immunostaining were anti-mouse labelled by Alexa 488 (1:1,000, ThermoFisher no. A28175) or Cy3 (1:1,000, Jackson ImmunoResearch Labs no. 115-165-146) and anti-chicken labelled by Alexa 647 (1:1,000, Jackson ImmunoResearch Labs, no. 103-605-155). The primary antibodies used in the western blots were mouse anti-GFP antibody (1:2,000, Roche, no. 11814460001), rabbit anti-mCherry antibody (1:2,000, Abcam, no. ab167453), rabbit anti-Ref2P antibody (1:500, Abcam, no. ab178440), rabbit anti-TMEM63A antibody (1:200, Novus Biologicals, no. NBP2-57359), mouse anti-GAPDH antibody (1:2,000, Proteintech, no. 60004-1-Ig) and mouse anti-tubulin antibody (1:2,000, Sigma, no. T9026). The secondary antibodies for the western blots were anti-mouse HRP antibody (1:4,000, Jackson ImmunoResearch Labs, no. 115-035-146) and anti-rabbit HRP antibody (1:4,000, Jackson ImmunoResearch Labs, no. 111-035-144).