Anti cd31 primary monoclonal antibody

Arteries were cut open longitudinally and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1 hr. Following a wash in PBS, arteries were permeabilized with 0.2% Triton X-100 and blocked with 3% BSA +5% serum for 1 hr at room temperature. For en face imaging experiments, arteries were incubated overnight with anti-PC-1 monoclonal primary antibody (E3 5F4A2, Baltimore PKD Center) and anti-CD31 primary monoclonal antibody (Abcam 7388) at 4°C. Arteries were then incubated with Alexa Fluor 488 donkey anti-rat, Alexa Fluor 546 donkey anti-mouse secondary antibodies (1:500; Molecular Probes), and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (1:1000; Thermo Fisher Scientific) for 1 hr at room temperature. After washing with PBS, arteries were mounted in 80% glycerol solution. DAPI, Alexa 488, and Alexa 546 were excited at 405, 488, and 561 nm with emission collected at ≤460 nm and ≥500 nm, respectively, using a Zeiss LSM 710 laser-scanning confocal microscope.

Arteries were cut open longitudinally and fixed with 4% paraformaldehyde in PBS for 1 hr. Following a wash in PBS, arteries were permeabilized with 0.2% Triton X-100 and blocked with 3% BSA +5% serum for 1 hr at room temperature. Arteries were incubated overnight with anti-PC-1 monoclonal primary antibody (E3 5F4A2, Baltimore PKD Center), anti-PC-2 monoclonal antibody (3374 CT-1 414, Baltimore PKD Center), and anti-CD31 primary monoclonal antibody (Abcam 7388) at 4°C. Arteries were then incubated with Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 546 donkey anti-rabbit secondary antibodies, and Alexa Fluor 647 goat anti-rat secondary antibodies (1:500; Molecular Probes) for 1 hr at room temperature. After washing with PBS, arteries were mounted in 80% glycerol solution. Lattice-SIM images were acquired on a Zeiss Elyra 7 AxioObserver microscope equipped with a 63× Plan-Apochromat (NA 1.46) oil immersion lens and an sCMOS camera. Lattice-SIM reconstruction was performed using the SIM processing Tool of Zeiss ZEN Black 3.0 SR software. Colocalization analysis of Lattice-SIM data was performed using Pearson’s and Mander’s coefficients.