Mab1d4

Brains were dissected in cold PBS 1X pH 7.4, fixed in 4% formaldehyde/PBT (PBS with 0.1% Triton X-100) for 25 minutes, then washed three times 15 minutes in PBT, and incubated overnight at 4°C with 0.3% Triton X-100 and 1% BSA. Samples were then incubated overnight at 4°C with primary antibodies in PBT, washed in PBT, and incubated overnight with secondary antibodies (4°C). After three washes in PBT, samples were mounted for analysis with Zeiss LSM 510 META or Leica SPE confocal microscopes. The following primary antibodies were used: mouse anti-FasII antibody (1:15; mAb1D4 from Developmental Studies Hybridoma Bank); rabbit anti-Hrp48 antibody (1:400; gift from D. Rio); rabbit anti-GFP antibody (1:1000; A1222, Molecular Probes). Cy3- or Cy5-coupled secondary antibodies (Jackson) were used at 1:1000.

Third instar larvae and 72- to 120-hr pupae were staged as described (Andres and Thummel 1994 (link); Bainbridge and Bownes 1981 (link)). The nervous systems of pupae and 0- to 5-d-old adults were dissected, fixed in 4% paraformaldehyde, and processed using standard protocols (Lee and Luo 1999 (link)). mAb1D4 (Van Vactor et al. 1993 (link)) (anti-Fasciclin II; anti-Fas II; 1:10) and mAb9.4A (Awasaki et al. 2000 (link)) (anti-Trio; 1:4) were obtained from the Developmental Studies Hybridoma Bank. The rabbit anti-Fas II (1:3000) was a gift from Vivian Budnik (University of Massachusetts). The rabbit anti-crustacean cardioactive peptide (anti-CCAP; 1:10,000) was a gift from John Ewer (University of Valparaiso, Chilé). Biotinylated anti-mouse and anti-rabbit IgG (1:200) were obtained from Vector Labs (cat. No. BA-9200 and BA-1000, respectively). Streptavidin Alexa Fluor 488, 546, and 568 (1:200) were obtained from Invitrogen (cat. No. S11223, S11225, and S11226, respectively). Preparations were imaged by confocal laser scanning microscopy using a Zeiss LSM 710 confocal microscope. Images were processed using ImageJ 1.46j (National Institutes of Health) and Photoshop CS5, and InDesign CS5 (Adobe).