Lsm 880 axio observer laser scanning confocal microscope

Nile Red (NR) staining (Sigma-Aldrich) and imaging of LDs was performed as described by Kokabi et al. (2019) (link). For the immunofluorescence detection of LiATG8, the cells were collected by centrifugation (3,000 rpm for 1 min) from approximately 2-ml aliquots of regularly diluted culture grown in the replete medium. The pellet was fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature (RT), and then rinsed three times with PBS. The cells were permeabilized with 1% Triton X-100 in PBS for 10 min at RT. After blocking with 2% bovine serum albumin in PBS for 30 min, cells were labeled at RT for 1 h with anti-ATG8 antibody diluted to 1:200 in a blocking solution. The cells were rinsed three times with PBS and incubated with secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG (Jackson IR Laboratories) at a dilution of 1:500 for 40 min. After washing three times, cells were observed under a Zeiss LSM 880 Axio Observer laser scanning confocal microscope using an alpha Plan-Apochromat 63×/1.4 Oil DIC M27 objective. Images were analyzed by Zen Blue software (Zeiss).