Mouse anti bruchpilot

Age-matched control and mutant 3^rd instar larvae were dissected in ice-cold Ca²⁺-free HL3.1 saline (Feng et al., 2004 (link)) and fixed for 30 min in HL3.1 containing 4% formaldehyde. Samples were washed in PBS followed by PBX (0.3% Triton X-100 in PBS) before antibody staining. Samples were mounted in either Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) or Prolong Gold antifade reagent (Life Technologies, Grand Island, NY, USA). The primary antibodies used include: mouse anti-Bruchpilot, 1:250 (Wagh et al., 2006 (link), obtained from Developmental Studies Hybridoma Bank (DSHB), The Univ. of Iowa, Iowa, USA); rabbit anti-GluRIII, 1:1000 (Marrus, 2004 (link)); mouse anti-α-spectrin, 1:50 (DSHB); rabbit anti-β-spectrin, 1:500 (gift from R. Dubreuil); mouse anti-DLG, 1:1000 (DSHB); mouse anti-adducin, 1:50 (DSHB); DyLight 649 conjugated anti-horseradish peroxidase, 1:4000 (Jackson Immunoresearch, West Grove, PA, USA). Secondary antibodies were conjugated to Alexa-488 or Rhodamine Red-X (Jackson Immunoresearch). Alexa 488 or Rhodamine conjugated phalloidin was used to stain F-actin (Life Technologies).

Brains and ventral nervous systems were dissected and stained using published methods ^{74 –76 (link)}. Antibodies used were rabbit anti-GFP (1:500, Invitrogen, #A11122), mouse anti-Bruchpilot (1:50, Developmental Studies Hybridoma Bank, University of Iowa, mAb nc82), Alexa Fluor 488-goat anti-rabbit (1:500, ThermoFisher A11034), and Alexa Fluor 568-goat anti-mouse (1:500, ThermoFisher A11031). Serial optical sections were obtained at 1μm intervals on a Zeiss 700 confocal with a Plan-Apochromat 20x/0.8NA objective. A detailed description of the staining and screening protocol is available at https://www.janelia.org/project-team/flylight/protocols under “IHC - Adult Split Screen.”