Rat α elav

Staining of adult and pupal brains was performed as previously described [18 (link),24 (link)]. Primary antibodies were used in the following dilutions: rabbit α-GFP 1:1,000 (Invitrogen), rat α-Ncad 1:20, rat α-elav 1:100, mouse α-Bruchpilot 1:20 (Developmental Studies Hybridoma Bank), mouse α-CD2 1:1,000 (Serotec). The following secondary antibodies were used: goat α-rabbit-FITC 1:1000, goat α-mouse-Cy3 1:100, goat α-rat-Cy3 1:200, goat α-guinea pig-Cy3 1:500, goat α-rat-Cy5 1:200, goat α-rabbit-Cy5 1:500 (Jackson ImmunoResearch), Alexa 568 goat α-mouse IgG highly cross-adsorbed 1:300 (Invitrogen). Confocal images were taken using an Olympus Fluoview FV1000.

Following dechorionation with a commercial bleach solution, embryos from overnight collections were devitellinized and fixed in a 1:1 mixture of heptane and 36% formaldehyde for 5 minutes and then washed in methanol. Embryos were then stored at -20 C or rehydrated, and used for staining. Primary antibodies used were: rat α-Elav 1:100, mouse α-BP102 1:100, mouse α-Eve 1:100 (Developmental Studies Hybridoma Bank, Indiana, USA); α-activated Caspase 3 1:100 (Cell Signaling, USA); and rat α-Deadpan 1:2, a gift from Cheng-Yu Lee. Secondary antibodies used were: Alexa flour 546 α-rat 1:100 (Santa Cruz Biotechnology, USA), Cy5 α-mouse 1:1000, Cy3 α-mouse 1:1000, FITC α-rabbit 1:1000 (Zymax, USA). Signal from α-deadpan staining was increased with the ABC kit from Vectastain (USA). A 510 Meta and 780 Duo confocal microscopes (Zeis, Germany) were used for fluorescent imaging, and images were processed with Zeiss software and ImageJ. Homozygous mutant embryos were selected by lack of TM3GFP of TM3LacZ. Accumulation of GFP::Fer1HCH in Sec23 mutants was evaluated using the fire LUT of ImageJ.

Larval filet preps were fixed in Bouin’s solution (a 1:5:15 ratio of acetic acid/formalin/picric acid) for 10 minutes at room temperature (VGLUT staining) or 4% paraformaldehyde in PBS for 20 minutes at room temperature (GFP staining) [56 (link)]. Blocking and staining were performed in PBS + 0.1% Triton X-100 +5% goat serum. The rabbit α-DVGLUT antibody was described previously (Daniels et al. [68 (link)]), and used at 1:5,000 dilution. Additionally, rat α-Elav (1:50, Developmental Studies Hybridoma Bank AB_52818; mouse α-beta galactosidase (1:100, Developmental Studies Hybridoma Bank AB_2314509). Other antibodies were: Cy3 and Cy5 conjugated goat α-HRP (1:1000, Jackson ImmunoResearch), Alexa 488 conjugated α-rabbit (1:1000, Molecular Probes) and Cy3 α-mouse (1:1000, Jackson ImmunoResearch), Alexa 488 conjugated Rabbit αGFP (1:1000 Life Technologies), AlexaFluor 488 chicken α-rat (1:1000, ThermosScientific-Invitrogen Cat# A-21470), Cy3 goat α-mouse (1:1000, Jackson ImmunoResearch, Cat#115-165-146), anti-HA (C29F4 Rabbit mAb, 1:1500 Cell Signaling). All samples were mounted and imaged in 70% glycerol containing Vectashield.