Lsm 800 confocal laser

All the confocal imaging was performed using a Zeiss LSM 800 confocal laser scanning upright microscope with a 40× long-distance working, water-dipping objective (W Plan-Apochromat 40x/1.0 DIC VIS-IRM27). Excitation was achieved using a diode laser with 488 nm for YFP and GFP, and 561 nm for TdTomato. The emission was collected at 500–550 nm for GFP, at 490–520 nm for YFP/Venus, and at 600–660 nm for TdTomato. For each reporter line all images were acquired with the same microscopy settings. Confocal stacks were acquired using a step size of 0.5–1 µm distance in z-dimension, at 16 bits image depth, and 512 × 512 pixel resolution. For samples that were larger than the microscope field of view, multiple overlapping stacks were acquired and stitched using MorphoGraphX^{34 (link),65 (link)}.

Cells were separated and seeded on coverslips in a 48-well plate. After cell adherence, the cells were fixed with paraformaldehyde (4%) and permeabilized with Triton X-100 (0.2%). The cells were then incubated with specific antibodies, counterstained with DAPI (0.2 mg/ml), and imaged under a Carl Zeiss LSM800 confocal laser scanning microscope.

The open reading frame of the IcSRO1 gene without the termination codon was amplified with specific primers (Table S1) and inserted into the pCambia-1300 expression vector containing the yellow fluorescent protein gene (YFP) under the control of CaMV35S promoter and NOS terminator. The empty vector containing only YFP sequence was used as the control. Leaves of one-week-old seedlings were harvested and the protoplast transformation was conducted according to the method reported by Yoo et al. [54 (link)]. After the transformation, the protoplasts were incubated at 23 °C in an incubator. After 12 h of incubation, a Zeiss LSM 800 confocal laser (Carl Zeiss, Jena, Germany) scanning microscope was used to observe the subcellular localization of YFP fusion protein. The excitation wavelength was 514 nm and the emission wavelength was 530–600 nm. The chlorophyll auto-fluorescence was recorded simultaneously.