Lsm 900 laser scanning microscope

For immunofluorescence microscopy, cells were grown on coverslips coated with PLO/laminin. Cells were washed with phosphate-buffered saline (PBS) with calcium and magnesium (PBS+/+) and fixed with 4% paraformaldehyde (PFA) for 10 min on ice. Subsequently, cells were washed with PBS+/+ and incubated with permeabilization buffer containing 0.1% Triton X-100 in PBS for 10 min on ice. After a washing step with PBS+/+, cells were incubated with 1% normal goat serum (NGS, Santa Clara, CA, USA) for 1 h at room temperature, followed by incubation with primary antibodies in 3% NGS at 4 °C overnight. The antibody used was mouse anti-Tom20 (1:100, Santa Cruz Biotechnology). Next, cells were washed with PBS+/+, followed by incubation with antimouse IgG conjugated with the Alexa488 secondary antibody (Invitrogen, Carlsbad, CA, USA) diluted 1:100 for 2 h at room temperature. After washing with PBS+/+, nuclei were stained with DAPI (5 min, 250 ng/mL). Cover slips were mounted using Fluoromount-G^® (SouthernBiotech, Birmingham, AL, USA). Microscopy was performed using an LSM 900 laser scanning microscope (LSM) equipped with a 130 × 100 STEP motorized scanning stage; a URGB laser module with a 405 nm, 488 nm, and 561 nm diode laser; a Plan-APOCHROMAT 63×/1.4 oil objective, and a GaAsP-PMT detector, using the ZEN 2 blue edition imaging software (all Zeiss, Hamburg, Germany).

The transformants expressing fluorescently labeled target proteins were cultured in V8 liquid medium for 48 h or inoculated into etiolated seedlings for 2 h (during the infection stage) and observed using LSM 900 laser scanning microscope (Carl Zeiss, Germany) at specific excitation and emission wavelengths (excitation wavelengths: GFP, 488 nm; mCherry, 561 nm; and BFP, 610 nm).

Root meristems were imaged by a Zeiss LSM 900 laser scanning microscope with a 20× objective. For confocal laser scanning microscopy, root meristems were mounted in 10 μg/mL propidium iodide. The process was performed according to the method described by Tian et al. [44 (link)]. In addition, to determine the number of cells belonging to the root meristem, root meristematic cortex cells were counted in a file extending from the QC to the first elongated cell excluded [46 (link)]. We quantified root cortical cell length in the maturation zone which has root hairs using 20 to 50 cells from 15 to 20 roots for each genetic background with Image J. Image processing was performed with the LSM image-processing software (Zeiss, Jena, Germany). We determined statistical significance by Student’s t test or one-way ANOVA (Tukey’s multiple comparison tests).