Calcoflour white stain

Calcoflour White Stain (Sigma Aldrich, St Louis MO) was prepared by mixing Calcoflour White Stain with 10% Potassium Hydroxide at 1/1 (v/v) and specimens were soaked under the coverslip in solution for 1 min prior to imaging. Specimens were imaged under an Olympus FV1200 Laser Scanning Microscope at 60×. All images were captured using Fluoview software version 4.2 with the same settings: excitation wavelength of 405 nm, dichroic beam splitter of 405/488/559 nm, and a bright field range of 70 nm starting at 410 nm. Minimal processing was performed aside from fluorescence normalization. The figure was cropped and edited in Adobe Photoshop and Illustrator.

Primary roots of Arabidopsis seedlings grown on vertically placed agar plates were photographed every day after 5–10 days of germination with a conventional image scanner (EPSON GT-S650) and the root length was analyzed by a FIJI/ImageJ software. To analyze cell shapes, primary roots of light-grown 4-day-old seedlings were stained with propidium iodide at 10 µg mL⁻¹ and observed with a confocal microscope Leica SP8 with a ×20, 0.75 NA HC PL APO CS2 objective (Leica). To analyze root cortex cells of the differentiation region, light-grown 4-day-old seedlings were fixed with 4% formaldehyde for 1 h, followed by several washes with the 1× phosphate-buffered saline, and then stained with a 1 : 5 diluted solution of Calcoflour White Stain (Sigma-Aldrich, #18909) for 30 min. After washing out an excess staining solution, root samples were mounted with the ClearSee solution for deep tissue imaging^{48 (link)}. Images were taken as 0.7 µm-step z-stacks with a Zeiss inverted microscope Axio Observer equipped with a confocal system LSM800 and a ×40, 1.1 NA LD C-Apochromat water-immersion objective (Zeiss). The root differentiation zone was determined by the proto-xylem differentiation