Dylight 594 goat anti rabbit

Animals were perfused with saline followed by 4% paraformaldehyde (PFA; Fisher Scientific) in 0.12 m PBS, at 4°C and pH 7.4. Brains were postfixed in PFA with 30% sucrose for 1 h and then transferred to 30% sucrose (Sigma-Aldrich) in PBS for 18 h at 4°C. Coronal slices (40 μm) were cut on a freezing microtome. Free-floating sections were incubated in rabbit anti-parvalbumin (1:2000 working dilution; Swant; RRID:AB_10000344) in PBS and 0.3% Triton X (Sigma-Aldrich) for 36 h at 4°C. Sections were washed three times for 10 min each in PBS before they were incubated in secondary DyLight 594 Goat Anti-rabbit (1:1000 working dilution; Jackson ImmunoResearch) in PBS and 0.3% Triton X. Sections were washed, mounted, and coverslipped using Prolong Gold Antifade with DAPI (Thermo Fisher Scientific). For each animal, a minimum of four sections including the prelimbic and infralimbic regions of the PFC were imaged on a confocal microscope (FluoView 1000, Olympus) at 20× magnification. The number of PV⁺ cells were hand counted in ImageJ (National Institutes of Health), and DAPI-labeled cells were counted using the thresholding function in ImageJ to obtain the percentage of total PV⁺ cells among all DAPI-labeled cells. PV expression in the treatment groups was normalized to the saline-treated control group to calculate the percentage change in PV.

We verified the specificity of GCaMP6f expression to dopaminergic structures by comparing direct eGFP fluorescence to immunoreactivity to tyrosine hydroxylase (TH-ir). Acute slices of midbrain and striatum were fixed overnight at 4 °C in 4% paraformaldehyde dissolved in PBS, then stored in PBS. After resectioning to 40 µm, free-floating sections were washed in PBS 5 × 5 min and incubated in 0.5% Triton X-100, 10% normal goat serum and 10% foetal bovine serum for 30 min. Slices were subsequently incubated overnight with 1:2000 primary (rabbit anti-TH; Sigma) antibody dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum and 1% foetal bovine serum. Sections were then washed with PBS 5 × 5 min and incubated for 2 h at room temperature with 1:1000 secondary (DyLight 594 goat anti-rabbit; Jackson) antibody dissolved in PBS containing 0.5% Triton X-100, 1% normal goat serum and 1% foetal bovine serum. Sections were washed with PBS and mounted on gelled slides with Vectashield mounting medium (Vector Labs) and imaged using a Zeiss LSM880 (confocal) running Zen black version 2.3, at 20 ×, N.A. 0.8. Maximum intensity projection from a z-stack of height 30 µm was captured individually and the stack of the pictures were compressed. TH (red) was captured at 638–759 nm with 633 nm excitation light. GCaMP (green) was excited with 488 nm and captured at 493–630 nm.