Ab153

Sections containing either mPFC (15 sections per animal) or the VTA (8 sections per animal) region were processed immunohistochemically for light microscope observation of the injection site and retrograde tracing, respectively. They were first rinsed in 0.1 M PB and incubated 30 minutes in 0.5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, CA) in 0.1 M PB to minimize non-specific background staining. They were then incubated overnight in rabbit anti-FG antibody (1:4000; AB153; Chemicon, Temecula, CA), 0.1% BSA and 0.25% Triton X-100 in 0.1 M PB protected from light at room temperature. After this, the sections were incubated in biotinylated goat anti-rabbit IgG (1:400; AP132B; Chemicon, Temecula, CA) and in 0.1% BSA in 0.1 M PB for 30 minutes, rinsed in 0.1 M PB and incubated 1 hour in avidin-biotin peroxidase complex (1:100; ABC; Vector Lab, Burlingame, CA). Peroxidase was visualized as a brown precipitate by incubation of the tissue in 0.022% 3,3´-diaminobenzidine (DAB) and 0.0033% hydrogen peroxide in 0.1 M PB at 4°C for 6–10 minutes.

One of the tissue series was processed for immunofluorescent detection of the retrograde tracer FG and of tyrosine-hydroxylase (TH, marker for dopamine-containing neurons in the VTA). All procedures were done protecting the tissue from light. After extensive rinsing in 0.1 M phosphate-buffered saline pH 7.4 (PBS), the sections were incubated in citrate buffer pH 6.0 at 90°C for 10 minutes. Then the sections were incubated in 10% donkey serum, 1% BSA and 1% Triton X-100 in 0.1 M PBS for 2 hours at room temperature. Afterwards, tissue sections were incubated in 1) rabbit anti-FG (1:1500; AB153; Chemicon, Temecula, CA) and 2) mouse anti-TH (1:1000; 22941; ImmunoStar Inc, Hudson, WI) in 3% donkey serum, 0.3% BSA and 0.3% Triton X-100 in 0.1 M PBS at 4°C for 48 hours. Next, the sections were rinsed in 0.1 M PBS and incubated in donkey anti-rabbit IgG 488 nm (1:100; A21206; Alexa Fluor, Invitrogen, Carlsbad, CA) and donkey anti-mouse IgG 570 nm (1:100; 715-025-150; Jackson ImmunoResearch, West Grove, PA) in 0.1 M PBS at 4°C for 2 hours.

The procedures were described previously^{37 (link),62} . Deeply anesthetized mice (ketamine, 90 mg/kg and Xylazine, 10 mg/kg) were perfused transcardially with 0.01 M PBS (PH 7.4) and paraformaldehyde (PFA) (4% in PBS). Spinal cord and brain were removed and post-fixed in 4% PFA for 2–4 h. The tissues were then cryoprotected in 20% sucrose overnight at 4 °C. Free-floating frozen sections were incubated with 2% donkey serum and 0.3% Triton X-100 for 1 h at room temperature followed by incubation with primary antibodies overnight at 4 °C. The sections were then washed and incubated with secondary antibodies for 2 h at room temperature. The following primary antibodies were used: chicken anti-GFP (1:500, Aves Labs, GFP-1020), guinea-pig anti-NK1R (1:500, AB15810, EMD Millipore), rabbit anti-FG (1:5000, AB153, Millipore). The following secondary antibodies were used: Alexa-Fluor 488 conjugated donkey anti-chicken (1:1000, Jackson ImmunoResearch, 703–545–155), Cy3-conjugated donkey anti-rabbit (1:1000, Jackson ImmunoResearch, 711–165–152) and Cy5-conjugated donkey anti-guinea pig 1(:1000, Jackson ImmunoResearch, 703–175–148). Fluorescent Images were taken using a Nikon C2+ confocal microscope system (Nikon Instruments, Inc.).