Aqua poly mount solution

Samples were permeabilized and blocked in incubation buffer (0.25 % Triton X-100 and 1% bovine serum albumin in PBS) for 60 minutes. Samples were then treated with primary antibodies (see Table S1) in incubation buffer either at 4°C overnight or four hours at room temperature. Samples were rinsed with wash buffer (0.05% Triton X-100 in PBS; 2 x 60 minutes) and treated with incubation buffer at least 60 additional minutes at room temperature. Samples were treated with 1:200 secondary antibodies (Alexa-conjugated, Thermo Fisher Scientific, USA) and 5μg/mL DAPI (4′6-Diamidino-2-Phenylindole Dihydrochloride, MP Biomedicals, 157574) in incubation buffer overnight at 4°C or at least two hours at room temperature. Samples were mounted by adding a drop of Aqua Polymount solution (Polysciences, Inc.) to the well and placing a round glass coverslip over the top. The mounted samples were stored overnight at 4°C and then sealed around the edges with fingernail sealant until imaging.
Confocal immunofluorescence microscopy images were collected using a Nikon A1R laser scanning confocal microscope with Plan Apo 10x, Plan Fluor 20x Ph1 DLL, or Plan Apo 20x DIC M objectives. Images were processed using NIS Elements and ImageJ. Some z-stacks were aligned using the “Align Current ND Document” (NIS Elements) before creating maximum projection images.

Panc-02 cells were grown on glass coverslips and fixed in ice-cold methanol and acetone for 20 min each. After 3 washes in PBS, cells were permeabilized with 0.2 % Triton X-100 in PBS for 1 h at room temperature. Nonspecific binding was blocked in PBS containing 5 % goat serum and 0.1 % Triton X-100 for 1 h. Cells were subsequently incubated in PBS with 5 % FBS, 0.05 % Triton X-100 with either a rabbit polyclonal CCK-A receptor antibody (PA-3116, Pierce, Rockford, IL, USA) or a CCK-B receptor antibody (#9491, CURE, Los Angeles, CA, USA) at a titers of 1:400 overnight at 4 °C. After three PBS washes, the cells were incubated in PBS containing 1 % FBS, 0.05 % Triton X-100, and a secondary goat anti-rabbit AlexaFluor 488-labeled antibody (Amersham Biosciences, Waltham, MA, USA) at a titer of 1:2,000 for 1 h at 4 °C in the dark. The slides were washed thrice with PBS, counterstained with Hoechst 33342 (0.5 μg/mL; AnaSpec, Fremont, CA, USA), and mounted with Aqua Poly/Mount solution (Polysciences, Warington, PA, USA). Images were captured on a Leica DM IRE2 confocal microscope.

P10 pups were deeply anesthetized with isoflurane and then perfused transcardially, first with PBS (pH 7.4) and with 4% paraformaldehyde (PFA) dissolved in PBS. The brains were removed and postfixed in 4% PFA overnight at 4°C, and then were coronally sectioned (40 μm thick) using a vibratome (Leica Biosystems). Tyrosine hydroxylase (TH) staining was accomplished as follows: (1) rinse 40 μm-thick slices in PBS (pH 7.4) three time; (2) block slices in 10% normal goat serum (Thermo Fisher, Cat. #: 50062Z) with 0.1% Triton X-100 (Sigma-Aldrich, Cat. #: T8787) in PBS for 1 h at room temperature; (3) incubate slices in rabbit polyclonal anti-TH primary antibody (1:700 in PBS; Sigma-Aldrich, Cat. #: T8700) for 24 h at 4°C (4) rinse slices in PBS three times; (5) incubate slices in Cy3-conjugated goat anti-rabbit IgG (1:100 in PBS, Jackson ImmunoResearch Laboratories, Cat. #: 111-165-003) and DAPI (1:1000) for 2 h at room temperature on a rotary shaker; (6) rinse slices in PBS three times and mount with an Aqua-Poly/Mount solution (Polysciences). TH-positive fibers in the cortex were imaged using a confocal microscope (Zeiss LSM800) and quantified by measuring the optical density of TH-immunofluorescence using MATLAB (Mathworks).