A1r confocal microscope

Cells grown on coverslips were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized in 0.1% Triton X-100/PBS for 5 min, and then blocked with 5% bovine serum albumin (BSA) in PBS containing 0.05% Triton X-100 for 1 h. Cells were incubated with appropriate primary and fluorophore-conjugated secondary antibodies for 2 h at room temperature (RT), washed, and then mounted with ProLong Antifade (Sigma).  For measuring the cell surface level of VEGFR2, VEGFR2 was stained in nonpermeabilized cells, using PBS blocking solution without having Triton in it. Cells were incubated with VEGFR2 primary antibody and fluorophore-conjugated secondary antibodies for 2 h at RT in the blocking buffer having no Triton, washed, and then mounted with ProLong Antifade (Sigma).  Images were obtained via a Nikon A1R confocal microscope or Zeiss Axio7/Apatome2 inverted fluorescence microscope with a monochrome camera.

After fixation, cells were stained for the cardiomyocyte marker TNNI3 (troponin I, cardiac 3; Millipore, MAB1691), nuclei (DAPI or DRAQ5; Thermo Fisher Scientific, D1306) and antibodies against the protein of interest (LC3B – Cell Signaling Technology, 2775; ZNF418 – Sigma-Aldrich, SAB2700695; FLAG – Sigma-Aldrich, F3165; ZFYVE1/DFCP1 – Cell Signaling Technology, 85156S; WIPI2 – Invitrogen, PAS-54,098). Slides were imaged using a Nikon Eclipse Ti inverted microscope with 10–20x air objective, Nikon A1R confocal microscope with 20–60x objective or a Zeiss Axiovert microscope with 40x or 63x oil objective and 7 images/well were captured. Images were analyzed with NIS Elements software.

Eyes were fixed for 15 min in 4% paraformaldehyde on ice and then rinsed twice with PBS. Upon fixing, the optic nerve and surrounding tissues were removed and the retinas were dissected with four radial incisions and then incubated in methanol at –20°C overnight. Methanol was rinsed out in PBS and then blocked (5% goat serum, 0.2% BSA, 0.3% Triton X-100 in 1× PBS) for 1 h at RT. Retinas were incubated with primary antibody in 100 µl of blocking solution at 4°C overnight and then washed for 15 min three times with 0.3% Triton X-100 in PBS. Retinas were incubated in secondary antibody at 4°C overnight and then washed four times for 30 min with 0.3% Triton X-100 in PBS. Retinas were mounted in anti-fade onto the cover slips and images acquired using a Nikon A1R confocal microscope or a Zeiss Axio7/Apatome2 inverted fluorescence microscope with a monochrome camera before analysis with the ImageJ/FIJI program.