Lsm 710 argon krypton laser scanning confocal microscope

Five-day-old seedlings were transplanted onto half-strength MS agar medium supplemented with various drugs for a 12-hour duration. Subsequently, the roots were stained with 5 μM propidium iodide (PI) for 15 seconds. The roots were mounted with sterile water and imaged using an LSM-710 argon/krypton laser scanning confocal microscope (Zeiss) with a 20 × objective. The excitation wavelengths for propidium iodide signals were set at 543 nm and emission was collected between 580 and 630 nm. Z-stack images were collected with 3 μm steps and the scan speed was 8 s/scan. The width of epidermis and cortex cells in the transition zone of the root was quantified using Image J software.

Subcellular localization assays were performed as previously described (Mao et al., 2014 (link)) with slight modifications. Briefly, 4-week-old Arabidopsis rosette leaves were digested by Cellulase R-10 and Macerozyme R-10 (Yakult Pharmaceutical) to prepare the mesophyll protoplasts. The protoplasts were resuspended with suspension solution (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, 2 mM 4-Morpholineethanesulfonic acid (MES) adjusted to pH 5.7 with KOH) and further transfected with 20 μg recombinant plasmid DNA (PROPEP1 to PROPEP8-pEZS-NL-GFP) by using polyethylene glycol-mediated transformation protocol (Sheen, 2001 (link)). The transformed protoplasts were incubated in the dark at 23°C for 16 h before confocal imaging analysis. Imaging was performed on an LSM-710 argon/krypton laser scanning confocal microscope (Zeiss) with a 63 × objective. FM 4-64 excitation at 514 nm and emission at 600-700 nm. GFP signals were excited at 488 nm wavelength and collected emission between 495 and 550 nm. Z-stack images were collected with 1 μm steps and the scan speed was 8 s/scan.