Immunostaining Methods for Drosophila Nervous System

Immunostaining methods were as previously described by Hewes et al. [33] (link). The CNS of the 90–100 hr (feeding IIIrd instar) AEL larvae were dissected in the standard saline that lacked calcium and fixed with 4% paraformaldehyde / 7% picric acid (v/v) in 1X PBS. Embryos were harvested from egg collection plates, dechorionated with 50% chlorine and fixed with 37% formaldehyde for 3 min in 50% heptane, then washed with 100% methanol. The primary antibodies used as peptide markers in this study are listed in Table 2. Anti-β-galatosidase antibody (Promega, WI, 1∶1000), MAb anti-neurotactin (BP106; Developmental Hybridoma Bank, Iowa City, 1∶100), MAb 4D9 anti-inv (gift from J. Skeath; 1∶10), rabbit anti-EVE (gift from J. Skeath; 1∶500), were also used. Antisera were raised to Drosophila DH 31, a kind gift from Julian Dow and to Drosophila Ast-B AWQSLQSSWamide (Research Genetics, Huntsville, AL). In both cases the peptides were coupled to porcine thyroglobulin using difluorodinitrobenzene as described by Tager [81] (link), in a ratio of 2 mg peptide to 5 mg carrier protein. Unreacted peptide was removed by dialysis and the conjugate injected in five to six sites on the back of a female New Zealand white rabbit. Booster injections were given at six week intervals. Blood was collected before the first injection and ten days after each booster injection; serum was collected and stored frozen. Cy3-conjugated, Alexa-568, Alexa-633 or Alex-488-conjugated, secondary antibodies were used at 1∶500 dilutions. Images were acquired on an Olympus FV500 laser scanning confocal microscope and manipulated by Adobe Photoshop software to adjust contrast. For the positional analysis of larval brain DIMM cells, the images acquired from the confocal microscope were imported into and analyzed with Amira software, as described at Pereanu and Hartenstein[48] (link).