Immunolabeling of Adult Male Brain

Adult male dissected brains were fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 25 minutes and labeled using a modification of protocols previously described [55 (link)]. The following primary antibodies were used: anti-bruchpilot (mAb nc82, 1:30, Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the Department of Biology, University of Iowa (Iowa City, IA).), monoclonal rabbit anti-GFP (1:200, Molecular Probes), rat anti-HA 3F10 (1:100, Roche), mAb dVGLUT (1:15), anti-TβH (1:400, [89 (link)]), rat anti-V5 (1:200, Biorbyt), and rabbit anti-TDC2 (1:100, Covalab). Secondary antibodies conjugated to Alexa 488, Alexa 594, or Alexa 647 (Molecular Probes) were used at a concentration of 1:200. Labeled brains were mounted in Vectashield (Vector Labs, #H1000). Images were collected on an Olympus Fluoview FV1000 laser scanning confocal mounted on an inverted IX81 microscope and processed using ImageJ (NIH) and Adobe Photoshop (Adobe, CA).