Alexa fluor 488 555

LSECs were seeded onto 12 mm micro coverglasses (Electron Microscopy Sciences). At different time-points (0 h, 24 h, 48 h), cells were fixed with 4% paraformaldehyde for 10min at room temperature, rinsed with PBS and permeabilized with 0.1% triton X-100 (Sigma) for 5 min. Thereafter, cells were blocked for 30 min with 1% BSA in PBS and consequently incubated with primary antibodies against LC3B (1:200, cell signaling) and Lamp2 (1:100, Santa Cruz) overnight at 4 °C.
Incubation with secondary antibodies conjugated with Alexa Fluor 488/555 (1:300, Invitrogen) was performed at room temperature for 1 h along with DAPI (3 ng/ml, Invitrogen). Preparations were then mounted using Fluoromount-G and dried overnight. Eight images per preparation and channel (visible; green, 488 nm; red, 555 nm) were obtained acquiring confocal z-stacks with a spectral confocal microscope (Leica TCS SPE). Images were then analyzed with the ImageJ software calculating the Pearson’s coefficient per cell. The Pearson’s correlation coefficient is a quantitative measurement that estimates the degree of overlap between fluorescence signals obtained in the 2 channels (green and red). The Pearson coefficients were averaged, and a standard error of the mean was calculated.^{22 (link)}