Cy3 tagged goat anti guinea pig antiserum

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.