Anti hrp tritc

All stocks were cultured on standard molasses/maize meal and agar medium in plastic vials or bottles at 25°C. Larvae were reared on apple juice plates supplemented with molasses/maize meal and yeast as previously described [79 (link)]. Larvae were selected for NMJ analysis at 5 days post egg laying. For analysis of bouton number was performed on the NMJ innervating muscles 6 and 7 from hemisegment A2 (1). Over 15 larvae were analysed for each genotype. For immunohistochemistry larvae were fixed for 20mins in 4% paraformaldehyde, or Bouin’s fixative for 30 minutes (GluRIIA). Primary antibodies used were anti-discs large (DLG, Developmental Studies Hybridoma Bank (DSHB), Iowa City, Iowa, USA),anti-Fas2 (DSHB), anti-GluRIIA (DSHB) and anti-BRP (DSHB), all used at 1/100. Secondary antibodies used were AlexaFluor 488 goat anti-rabbit and AlexaFluor 633 goat anti-mouse (Invitrogen) at 1/1000, and anti-HRP-TRITC (The Jackson Laboratory, Bar Harbor, Maine, USA). Z-stacks were taken using a laser-scanning confocal microscope (Leica TCS SP5 II confocal microscope) and analysis performed using ImageJ and Adobe Photoshop. For statistical analysis of the genetic interactions, ANOVA was performed between the control, the two single heterozygous mutations and the transheterozygotes.

NMJs from third instar larvae and adult brains from flies of 1 week old without mouthparts were dissected and fixed with 4% formaldehyde in 1X PBS following standard protocols previously described. Reagents used include: Normal Donkey Serum (0.5%, NDS, Jackson Lab), mouse anti-Repo (1:100, DSHB), rat anti-Elav (1:500, DSHB), rabbit anti-Cnx (1:500, gift from Nansi Colley), mouse anti-Para (1:100, made in this study), anti-HRP-TRITC (1:500, Jackson Lab), and anti-HRP-Cy5 (1:500, Jackson Lab). All other secondary antibodies were purchased from Jackson Lab. Images were captured with Leica TCS SP5 confocal microscopy. Details on brain dissection, immunostaining, image acquisition, and quantification of fluorescent intensities were included in the supplementary materials.

Stage 12 (7–9 hours) and 15 embryos (11–13 hours) collected from Grape plates were dissected and fixed with 4% formaldehyde in 1X PBS following standard protocols previously described. Reagents used include: Normal Donkey Serum (0.5%, NDS, Jackson Immunoresearch), anti-HRP-TRITC (Jackson Immunoresearch, 1:500), mouse anti-Repo (DSHB, 1:100). All other secondary antibodies were purchased from Jackson Immunoresearch. Images were captured with Leica TCS SP5 confocal microscopy.