Goat anti rabbit alexa 546

The antibodies used were mouse anti-FLAG (Sigma), rabbit anti-Zhangfei serum [12 (link)], rabbit anti-Xbp1 (Abcam, Cambridge, MA), rabbit anti-HERP (Abcam, Cambridge, MA), rabbit anti-GRP78 (Abcam, Cambridge, MA), and mouse anti-GAPDH (Chemicon, Billerica, MA). Suppliers of antibodies against Xbp1, HERP, GRP78 and GAPDH indicated that they recognized canine proteins. Secondary antibodies were goat anti-mouse Alexa488, goat anti-rabbit Alexa546 and goat anti-rabbit Cy5 (Invitrogen). Cells were processed for immunoblotting and immunofluorescence as described previously [5 (link),12 (link)].

We used rabbit antisera to A-chains of DILP2 and DILP330 (link) and C-peptide of DILP531 (link) at a dilution of 1:2000. A rabbit antiserum to Drosophila prothoracicotropic hormone (PTTH68 (link)), was kindly provided by Dr. P. Leopold (Nice, France) and applied at 1:500. The central nervous system of third instar larvae, different pupal stages and adult CNS, as well as various tissues (fat body, gut, ovaries) were fixed in ice-cold 4% paraformaldehyde (4% PFA) in 0.1 M sodium phosphate buffer (PB; pH 7.4) for 2–4 h. After washing with PB 3 × 15 min, tissues were dissected in PB. All tissues were washed finally in 0.01 M phosphate buffered saline (PBS) with 0.25% Triton-X (PBS-Tx) for 15 min. Tissues were then incubated in primary antibodies for 24–48 h at 4 °C with gentle agitation. After washes (4 × 15 min) in PBS-Tx, secondary antibodies were applied for overnight or 48 h at 4 °C. Tissues were then washed in PBS-Tx 7 × 10 min, rinsed in 0.01 M PBS and mounted with 80% glycerol in 0.01 M PBS. Rabbit and mouse anti-GFP (1:1000) were used (Invitrogen, Carlsbad, CA). The following secondary antibodies (1:1000) were employed for detection: goat anti-rabbit Alexa 546, goat anti-rabbit Alexa 488, goat anti-mouse Alexa 488, goat anti-mouse Alexa 546 (all from Invitrogen), Cy3-tagged goat anti-guinea pig antiserum (Jackson ImmunoResearch, West Grove, PA).

For immunolabeling, tissues from larvae or female adults were dissected in chilled 0.1 M phosphate buffered saline (PBS). They were then fixed for 4 h in ice-cold 4% paraformaldehyde (PFA) in PBS, and subsequently rinsed in PBS three times for 1 h. Incubation with primary antiserum was performed for 48 h at 4°C with gentle agitation. After rinse in PBS with 0.25% Triton-X 100 (PBS-Tx) four times, the tissues were incubated with secondary antibody for 48 h at 4°C. After a thorough wash in PBS-Tx, tissues were mounted in 80% glycerol with 0.1 M PBS.
The following primary antisera were used: Rabbit or guinea pig antiserum to part of the C-peptide of DILP1 diluted 1:10,000 (17 (link)). Rabbit antisera to A-chains of DILP2 and DILP3 (36 (link)) and part of the C-peptide of DILP5 (37 (link)) all at a dilution of 1:2,000, mouse anti-green fluorescent protein (GFP) at 1:000 (RRID: AB_221568, Invitrogen, Carlsbad, CA). The following secondary antisera were used: goat anti-rabbit Alexa 546, goat anti-rabbit Alexa 488, and goat anti-mouse Alexa 488 (all from Invitrogen). Cy3-tagged goat anti-guinea pig antiserum (Jackson ImmunoResearch, West Grove, PA). All were used at a dilution of 1:1,000.

Adult flies were dissected in ice-cold Drosophila Ca²⁺ free saline. After removing wings and legs, brain-thoracic/abdominal ganglia complexes were quickly excised from head and thorax. All preparations were fixed overnight in Zamboni's fixative overnight at room temperature, washed and treated as described in detail earlier [29 (link)]. The only modifications concerned the use of two different primary and secondary antibodies always at the same time of incubations. Primary antibodies were a polyclonal rabbit anti-DrmITP diluted 1:10,000 [33 (link)] and a monoclonal mouse anti-GFP (against Jelly fish GFP; Invitrogen) diluted 1:1,000. Secondary antibodies were goat anti-rabbit Alexa 546 and goat anti-mouse Alexa 488, respectively (Invitrogen), both diluted 1:1,000. Preparations were imaged with a Zeiss LSM 780 confocal microscope by use of 10× or 20× objectives. Confocal images were processed with Zeiss ZEN software, version 8.1 2012, for maximum intensity projections of z-stacks. Brightness and contrast was adjusted using Corel Photopaint X7 during plate-mounting using Corel Draw X7.

Cells were processed as described before26 (link) and in supplementary material using a Leica SP5 confocal microscope (Leica Microsystems). The primary and secondary antibodies used in this work were obtained from the same batches and always utilized at the same dilutions. The following antibodies were used: rabbit anti-ASMase (Santa Cruz Biotechnologies, USA) with a dilution of 1:50 overnight in humid chamber at room temperature, rat anti-LAMP-2 (Hybridoma Bank, Iowa, USA) with a dilution of 1:50 during 2 h at room temperature, rabbit polyclonal antibody anti-sortilin (kindly provided by Dr. Claus Petersen, University of Aarhus, Denmark) with a dilution of 1:50 over night in humid chamber at room temperature. Mouse anti-EEA1 (BD Bioscience) with a dilution of 1:50 over night in humid chamber at room temperature, mouse anti-GM130 (BD Bioscience) used with a dilution 1:100, 2 h at room temperature and mouse anti-Syntaxin6 (BD Bioscience) with a dilution of 1:50 over night in humid chamber at room temperature. Secondary antibodies used for indirect immunofluorescence were: goat anti-rabbit Alexa633 (dilution 1:600), goat anti-mouse Alexa633 (dilution 1:600), goat anti-rabbit Alexa488 (dilution 1:600) and goat anti-rabbit Alexa546 (dilution 1:600) (Invitrogen, USA). Secondary antibodies goat anti-Rat Cy3 and goat anti-Rat Cy5 were from Jackson Research Inc, USA.