Ab13970

Brains were stained with the following primary antibodies: polyclonal goat anti-GFP fused with FITC (1:1,000, Abcam, ab6662), polyclonal rabbit anti-GFP (1:1,000, Molecular Probes, A6455), polyclonal chicken anti-GFP (1:1,000, Abcam, ab13970), monoclonal mouse anti-TH (1:500, ImmunoStar, 22941), polyclonal rabbit anti-TDC2 (1:200, CovalAb, pab0822-P), monoclonal mouse anti-ChAT (1:150, Developmental Studies Hybridoma Bank, ChaT4B1), polyclonal rabbit anti-GABA (1:500, Sigma, A2052), and rabbit anti-dVGlut (1:5,000)^{70 (link)} for identifying GFP-positive, dopaminergic, octopaminergic, cholinergic, GABAergic, and glutamatergic neurons. The following secondary antibodies were used: polyclonal goat anti-chicken Alexa Fluor 488 (1:200, Molecular Probes, A11039), polyclonal goat anti-rabbit Alexa Fluor 488 (1:200, Molecular Probes, A11008), polyclonal goat anti-rabbit Alexa Fluor 568 (1:200, Molecular Probes, A11011), polyclonal goat anti-rabbit Cy5 (1:200, Molecular Probes, A10523), polyclonal goat anti-mouse Alexa Fluor 647 (1:200, Molecular Probes, A21235), and polyclonal goat anti-rabbit Alexa Fluor 647 (1:200, Molecular Probes, A21245).

Mice were anesthetized with pentobarbital and transcardially perfused with a 4% formaldehyde/PBS solution. The brains were then extracted and post-fixed overnight before being stored in PBS for later sectioning. Forty micron thick sections were blocked in a 5% bovine serum albumin, 0.2% Triton X-100, PBS solution for 4 h at room temperature. The sections were then incubated in a primary antibody/PBS solution overnight as follows: chicken anti-GFP (1/ 2000; Abcam, ab13970), rabbit anti-MOR (1/4000; Immunostar, 24216), rabbit anti-D2DR (1/500; Frontier Institute, D2R-Rb-Af750), and rat anti-DAT (1/5000; Millipore, MAB369). Sections were then washed three times in 0.2% Triton X-100/PBS before being incubated in secondary antibody solutions with Alexa488 goat anti-chicken (1/2000), Alexa568 goat anti-rabbit (1/1000), or Alexa568 goat anti-rat (1/1000) overnight (secondary antibodies were from Life Technologies). Following five washes in PBS, sections were mounted onto subbed slides, coverslipped with Fluoromount G (Electron Microscopy Sciences), and imaged with a Zeiss Lumar stereoscope and an Axiovert 200 microscope equipped with DAPI, eGFP, and Cy3 filter sets and an Axiocam MR fluorescence camera with Axiovision software (Zeiss).

The primary antibodies used were: anti-GFP (1:1000, chicken, Abcam, catalog# ab13970, lot# GR236651-24); anti-TH (1:1000, rabbit, Immunostar, catalog# 22941, lot# 1552001); and anti-mCherry (1:1000, mouse, Living Colors/Clontech, catalog# 632543, lot# 1506199A). Fluorophore-conjugated secondary antibodies were purchased from ThermoFisher Scientific. Specifically, the following secondary antibodies were used in this study: goat anti-rabbit-488 (1:500, catalog# A11034, lot# 1737902), goat anti-chicken-488 (1:500, catalog# A11039, lot# 1759025) and goat anti-mouse (1:500, catalog# A11004, lot# 1698376). Antibodies were diluted in PBS with 10% NGS and PBST. Additional validation details are available from the manufacturer for each antibody used in this study.