Arteries were pinned to a six-well plate containing Sylgard and fixed in 4% paraformaldehyde for 20 min at room temperature. Arteries were washed three times in 100 μM glycine solution, followed by three washes with phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100 in PBS for 10 min, and washed again three times with PBS. Arteries were then incubated in antibody buffer solution in PBS for 1 hour at room temperature and then washed three times with antibody wash solution containing 5% bovine serum albumin (BSA). The arteries then were incubated with a rabbit anti-P2Y2 receptor antibody (1:100; sc-20124, Santa Cruz Biotechnology) and goat anti-CD31/PECAM-1 (1:200; AF3628, R&D Systems) in PBS containing 1% BSA and 2% Donkey serum for 12 hours at 4°C. Arteries were washed with antibody wash solution containing 5% BSA three times, followed by incubation with Alexa Fluor 488 donkey anti-rabbit antibody (Invitrogen) and Alexa Fluor 568 donkey anti-goat antibody (Invitrogen) in antibody buffer solution for 1 hour at room temperature. The P2Y2 receptor secondary antibody was an Alexa Fluor 488 donkey anti-rabbit antibody (1:1000) and excited using 488 nm. The CD31/ PECAM-1 secondary antibody was an Alexa Fluor 568 donkey anti-goat secondary antibody (1:1000) and was excited using 568 nm. Excitation light was provided by wide-field epifluorescence illumination via a monochomator (Photon Technology International/Horiba UK Ltd.) and visualized using a back-illuminated EMCCD camera (13-μm pixel size; iXon Life 888, Andor) through a 60× (water immersion; numerical aperture of 1.0; Nikon S Fluor) objective lens. Fluorescence emission was recorded at 10 Hz. Fluorescence illumination was controlled, and images (16-bit depth) were captured using ImageJ (National Institutes of Health, Bethesda, MD, USA). Negative controls were performed in the absence of primary antibody.

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