Ab125

Larval IHC was performed as previously published (Avants et al., 2011 (link)), using antibodies raised against total ERK (ERK; p44/42 MAPK [ERK1/2], #4696, Cell Signaling Inc, Danvers, MA, USA), Islet-1 and Islet-2 homeobox (Islet1/2, #39.4D5, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), TH1 (AB152, Sigma-Aldrich Inc, Burlington, MA, USA), and 5-HT (AB125, Sigma-Aldrich Inc). IHC-stained larvae were imaged on a Nikon A1R multiphoton microscope, using a water immersion 25×, NA 1.1 objective.

Drosophila larvae were fixed in 8% paraformaldehyde (Electron Microscopy Science, Hatfield, U.S.A.) in PBS at room temperature for 30 min. After blocking for 2 h in 1% BSA in PBS/0.1% Triton-X 100, inverted larvae were incubated overnight in primary antibody [anti-5HT (Sigma-Aldrich AB125, 1:2000), anti-shroud antibody (gift from R.Niwa) (1:500), anti-Syt1 antibody (1:100, Developmental Studies Hybridoma Bank). Primary antibody staining was detected using Alexa Fluor 488 (Molecular Probes) goat-anti rabbit secondary antibodies (1:5000). DNA was visualized by staining with Hoechst 33258 (Invitrogen, 1:10,000). Brains with attached prothoracic glands were then dissected out and mounted on coverslips using mounting media (Vectashield). Images were captured using a Zeiss confocal microscope LSM 880 or a Zeiss Observer. Z1 microscope.