A1r spectral scanning confocal microscope

CaCo2 cells were stained by using cell mask green plasma membrane stain (Thermo Fisher Scientific) following the manufacturer’s recommendations. Images of stained cells were obtained with a A1R spectral scanning confocal microscope (Nikon Co.) that incorporates a total internal reflection fluorescence (TIRF) module.

Mice were deeply anesthetized with pentobarbital (300 mg/kg i.p.) and transcardially perfused with 0.1M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA, Sigma). Brains were removed, post-fixed in 4% PFA for 24 h at 4°C, and cut in 50 μm sections on a vibratome. For tyrosine hydroxylase (TH) immunohistochemistry, brain sections were rinsed in PBS (0.1 M) and incubated for 1 h at room temperature in a blocking solution containing 5% bovine serum albumin (Sigma) and 0.3% Triton X-100 (Axon Lab AG) in PBS. Sections were then incubated overnight at +4°C with a rabbit anti-TH antibody (1:500; Millipore) in blocking solution, then rinsed in PBS, incubated for 2 h at room temperature with an alexa-conjugated goat anti-rabbit antibody (1:500; Invitrogen) in blocking solution. Sections were then rinsed again in PBS and mounted on glass slides with Fluoroshield Mounting Medium (Abcam). Confocal images were captured with a Nikon A1r Spectral scanning confocal microscope, and then processed with ImageJ.