Dcsp 3

Third instar larvae were dissected, fixed, and immunostained as described [25 (link),59 ]. Briefly, larvae were dissected in dissection buffer (2X stock contains 128 mM NaCl, 4 mM MgCl₂, 2 mM KCl, 5 mM HEPES, and 36 mM sucrose, pH 7.2). Following dissection, larvae were treated with 0, 10, or 50 mM EtOH at 25°C for 20 min. Larvae were fixed in 4% formaldehyde and incubated with primary antibodies against either rabbit monoclonal cysteine string protein (DCSP-3, 1:10, Developmental Studies Hybridoma Bank) overnight. As needed larvae were also incubated with the neuronal membrane marker Texas Red-conjugated horse radish peroxidase (HRP) and secondary antibody (Alexa anti-rabbit 488, 1:100, Invitrogen) for 2 hrs at room temperature, mounted using Vectashield mounting medium (Vector Labs). Images were obtained using a Nikon TE-2000E inverted microscope at 60×. At least 5-10 larvae were imaged from each genotype.

Third instar larvae were dissected, fixed, and immunostained as described (Fye et al 2010 , Gunawardena & Goldstein 2001 (link)). Briefly, larvae were dissected in dissection buffer (2X stock contains 128 mM NaCl, 4 mM MgCl₂, 2 mM KCl, 5 mM HEPES, and 36 mM sucrose, pH 7.2). Following dissection, larvae were treated with 0, 10, or 50 mM EtOH at 25°C for 20 min. Larvae were fixed in 4% formaldehyde and incubated with primary antibodies against either rabbit monoclonal cysteine string protein (DCSP-3, 1:10, Developmental Studies Hybridoma Bank; RRID:AB_528184) overnight. Larvae were incubated with the neuronal membrane marker Texas Red-conjugated horse radish peroxidase (HRP) and secondary antibody (Alexa anti-rabbit 488, 1:100, Invitrogen) for 2 hrs at room temperature, mounted using Vectashield mounting medium (Vector Labs) and imaged using a Nikon TE-2000E inverted microscope at 60X.