Ab154610

Protein extraction from MA was carried out by tissue immersion in ice-cooled acetone, 10% trichloroacetic acid and 10 mM dithiothreitol (DTT). Tissue was then lyophilized, disrupted and heated at 95°C for 10 minutes in SDS-gel sample buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 0.1 M DTT) [24 (link),25 (link)]. Finally, proteins were extracted by continuously mixing samples in SDS-gel sample buffer overnight at 4°C. Proteins were separated by SDS-PAGE on 7.5% acrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked using 5% fat-free dry milk and then incubated with primary antibodies (1:1000; PLCβ3 Abcam #ab52199; PLCδ1 Abcam #ab154610; PLCγ2 Abcam #ab18983) for 1 hour at room temperature. Membranes were then washed and incubated with HRP-conjugated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) for 1 hour at room temperature. Immunoreactive bands were revealed by enhanced chemiluminescence (Western Lightning Plus, PerkinElmer).

MA were cut longitudinally, and pinned (endothelium en face) on a Sylgard block. Arteries were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 then blocked with 4% normal donkey serum. Following overnight incubation at 4°C with primary antibodies (1:500; PLCβ3 Abcam #ab52199; PLCδ1 Abcam #ab154610; PLCγ2 Abcam #ab18983) in 0.1% Triton X-100, arteries were incubated for 1h with an Alexa Fluor 555-conjugated donkey anti-rabbit secondary antibody (Invitrogen). DAPI was applied to stain nuclei prior mounting for confocal imaging. Autofluorescence of the internal elastic lamina was also acquired. Fluorescence emissions were detected using a Zeiss LSM 510 confocal microscope (63X oil objective/1.4, ex: 405 nm, 488 nm and 543 nm). All images were deconvolved with Huygens professional software using experimentally determined point spread functions (PSFs) and reconstructed with Zen 2009 Light Edition.