Anti rat alexa 488 coupled igg

For IHC rats were deeply anesthetized with pentobarbital, intracardially perfused with 4% formalin, and the tissue processed with standard methods. Briefly, sections were incubated overnight in either mouse CamKII IgG (1:250; Chemicon) or mouse anti-GAD67 IgG (1:2000; Chemicon) and double-labeled with rat anti-D1 DAR IgG (1:250; Sigma), washed, and incubated for 60 min with anti-mouse TRITC coupled IgG (1:200; Molecular Probes) and anti-rat Alexa 488-coupled IgG (1:200; Molecular Probes). Sections were washed, mounted on slides, and imaged with confocal microscopy. Three regions of interest were drawn within the injection bolus (visualized with FITC filter). Within each region of interest (ROI), z-series stacks were generated in the FITC (D1 immunoreactivity [IR]) and TRITC (GAD67- or CamKII-IR) channels. Within each ROI the numbers of D1-labelled cells that were co-localized with GAD67, CamKII, or “other” (D1-immunoreacive) were recorded. Three ROIs were generated per section, with four serial sections analyzed (section thickness=40µm; section interval=400µm; total cells counted~360/animal). Absolute cell numbers across all three ROIs were reported.

Transduced rats were deeply anesthetized and intracardially perfused and the tissue was processed with standard methods (Sonntag et al., 2014 (link)). Briefly, sections were incubated overnight in rat anti-D1 DAR IgG (1:250; targeting the intracellular C-terminus; Sigma), washed, and incubated for 60 min with anti-rat Alexa 488-coupled IgG (1:200; Molecular Probes). GFP expressed sufficiently as to not warrant staining (images of our viruses can be found in (Sonntag et al., 2014 (link))). Sections were washed, mounted on slides, and placement verified through the use of a Zeiss AxioScop microscope. Only the data presented here had viral placements within the plPFC.