Mouse anti discs large

Flies, in which CrebB knockout was induced, were moved for 6 days to 29°C before antibody staining experiments. Male adult brains were dissected in phosphate-buffered-saline (PBS) and fixed at room temperature for 30 min with a 3.7% formaldehyde solution (in PBS). Brains were washed at least five times with PBST (PBS with 0.3% triton X-100) before primary antibodies were added for overnight incubation at 4°C. The following primary antibodies were used: rabbit anti-Eyeless (1:400, courtesy of Uwe Waldorf), chicken anti-GFP (1:1000, Abcam ab13970), rabbit anti-GFP (1:1000, Invitrogen A-6455), guinea pig anti-CrebB (1:400), rabbit anti-Tyrosine hydroxylase (1:100, Merck AB152), mouse anti-Repo (1: 20, Developmental Studies Hybridoma Bank 8D12), mouse anti-Discs large (1:50, Developmental Studies Hybridoma Bank 4F3). Brains were washed again before the overnight incubation at 4°C with secondary antibodies. The used secondary antibodies were conjugated with Alexa fluorescent proteins (488, 567 or 647; Molecular Probes) and diluted 1:200. After a last washing step, brains were mounted in Vectashield H-1000 or Vectashield with DAPI H-1200 (Vector Laboratories) on a microscope slide. Samples were imaged with Leica SP5 confocal microscope and the images were processed with Imaris Bitplane 9.2 and Adobe Photoshop CS6.

Larval tissues were dissected in PBS and fixed for 10 min in 3.7% formaldehyde in PBS containing 0.1% Triton X-100 and processed as previously described^{51 (link), 52 (link)}. The following primary antibodies were used: guinea pig anti-Deadpan (from J. A. Knoblich), mouse anti-Discs large, and rat anti-DEcad2 (from Developmental Studies Hybridoma Bank). Alexa-conjugated secondary antibodies (Invitrogen) were used. The nuclei were stained with Hoechst 33342 (Invitrogen). Images were acquired with a Zeiss LSM700 confocal microscope and were processed in Photoshop (Adobe Systems). Staining of acidic organelles was done as described previously^{8 (link)}.

Adult Drosophila brains were dissected in phosphate buffered saline (PBS) and collected in PBS; 1% bovine serum albumin (BSA) before fixing in a PBS; 4% formaldehyde; 4% sucrose solution for 30 minutes at 25°C. Similarly, wandering third instar larvae were dissected in PBS and fixed as above. Brains and larval preparations where then washed with PBS; 1% BSA; 0.2% Triton-X 100 three times for 30 minutes each at 25°C. All samples were then incubated in primary antibody for 14–16 hours at 4°C, washed three times in wash solution for 30 minutes each at 25°C, and then incubated in secondary antibody for 1 hour. Finally, samples were washed three times for 45 minutes each before mounting in Fluoromount G (eBioscience, Inc.). Primary antibodies used: mouse anti-Cbp53E (1:50, DCB32(pok13), Developmental Studies Hybridoma Bank)[37 (link)], guinea pig anti-insulin (1:50, ab7842, Abcam), rabbit anti-horseradish peroxidase (1:200, Jackson Laboratories), mouse anti-Discs Large (1:200, 4F3, Developmental Studies Hybridoma Bank). Secondary antibodies used (all at 1:2000 dilutions): AlexaFluor 647 goat anti-mouse (Invitrogen), AlexaFluor 488 goat anti-rabbit (Invitrogen), AlexaFluor 488 goat anti-mouse (Jackson Laboratories), and Cy3 goat anti-guinea pig (Invitrogen).