Elyra 7 axioobserver microscope

Lattice-SIM images were acquired on a Zeiss Elyra 7 AxioObserver microscope equipped with an Plan-Apochromat 63×/1.4 Oil DIC M27 objective and two pco.edge sCMOS (version 4.2 CL HS) cameras. The system contained 405 nm and 642 nm diode, and 488 nm and 561 nm OPSL lasers. For each focal plane 13 phase images were acquired. Exposure time and laser power were balanced for each fluorescence channel individually to minimize bleaching and exposure time. SIM and Lattice-SIM reconstruction was performed with the SIM processing Tool of the ZEN 3.0 SR (black) software.
Information on the specific hardware configurations and acquisition settings of all microscopy platforms are available on request.

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.