Tcs sp8

Before choline treatment, choline chloride was dissolved in water to make a 100-mM choline solution. In the experiment, the choline solution was diluted to a concentration of 1 mM, and the seedlings were treated for the desired time.
For BFA treatment, 6-day-old Arabidopsis seedlings were immersed in 50 μM BFA for 1.5 hours, and then the BFA bodies were observed by confocal laser scanning microscopy (Leica TCS SP8). In the washout experiments, the BFA-treated seedlings were incubated in water for 2 hours, after which the BFA bodies were examined by confocal laser scanning microscopy (Leica TCS SP8).
For CHX and BFA treatment, 6-day-old Arabidopsis seedlings were pretreated with 50 μM CHX for 30 minutes, followed by being immersed in a solution with 50 μM CHX and 50 μM BFA for 1.5 hours, and then the BFA bodies were observed by confocal laser scanning microscopy (Leica TCS SP8). In the washout experiments, above seedlings were incubated in water for 2 hours, and the BFA bodies were then examined by confocal laser scanning microscopy (Leica TCS SP8).
For 1-butanol treatment, the Arabidopsis seeds were grown on 1/2 MS salt medium plate for six days, and the seedlings were then transplanted to the 1/2 MS salt medium plate containing 0.4% 1-butanol and grew for another five days. The root length was measured by Image J.

U373 cells were seeded on poly-L-lysine (Sigma-Aldrich, P6282-5MG)-coated coverslips in 6-well plates, and Oil red O staining was performed as previously reported [60 (link)]. Briefly, cells were fixed in paraformaldehyde (4% solution) for 10 min and gently rinsed three times with PBS. Fixed cells were incubated with 60% isopropanol for 5 min and then washed with distilled water. Subsequently, cells were probed with 1 mL of Oil Red O working solution (Sigma-Aldrich, O1391-250ML) for 15 min at room temperature by putting the 6-well plate onto an orbital rotator shaker. After the incubation with the staining solution, wells were washed three times with distilled water to eliminate the excess stain. Coverslips were finally mounted with Fluoroshield mounting medium (Sigma-Aldrich, F6182), and Oil red O autofluorescence was visualized via confocal microscopy (TCS SP8; Leica, Wetzlar, Germany). Images were captured using Leica TCS SP8 equipped with a 63× magnification and Leica LAS X Software (Milan, Italy). Quantification of Oil red O staining was performed through ImageJ software for Windows and calculated as mean fluorescence intensity per cell area.

Small pieces of young leaves from A. thaliana or N. benthamiana were mounted in water between a slide and a coverslip and were immediately observed. Live-cell imaging was performed on Leica TCS SP8 confocal laser scanning microscope (Leica Microsystems) with a 40X/1.40 oil immersion objective. Images were taken at 1024 × 1024 pixels resolution using line-by-line sequential scanning (when appropriate). The excitation wavelength for eGFP was 488 nm and its emission was collected from 500 to 525 nm. Z-stacks of between 50 and 100 confocal images were acquired and used to generate three-dimensional (3-D) reconstructions using Leica TCS SP8 software (when required). The LAS AF Lite software (Version 3.3) and Adobe Photoshop CS6 were used for the post-acquisition images processing.