Confocal microscopy was performed on a Zeiss LSM700 and a Leica TCS SP8 confocal laser scanning microscope as previously reported (Kim et al., 2020 (link); Seo and Lee, 2021 (link)). Pictures were taken with either a 20× dry objective lens or a 63× water-immersion objective lens. For Z-scan imaging, the same position on the root and the same laser scanning area were chosen.
To image the ProTCSn::ntdTomato, ProDR5v2::n3GFP dual-marker line, seedlings were fixed in 4% paraformaldehyde and treated with ClearSee solution (Kurihara et al., 2015 (link)) for five days. The seedlings were then stained with 0.1% (v/v) calcofluor white 2MR (Sigma-Aldrich, USA) in ClearSee solution for 30 min and observed under a confocal microscope (Leica TCS SP8) with preset excitation/emission wavelengths of 488 nm/500-530 nm for GFP, 550 nm/580 nm for tdTomato and 350 nm/420 nm for calcofluor white 2MR. To visualize the GFP protein in other transgenic lines, seedlings were stained in a 1 μM propidium iodide (PI; Sigma-Aldrich, USA) solution for 2 min and observed under a Carl Zeiss LSM700 confocal microscope (Carl Zeiss, Germany) with the following excitation and detection windows: GFP 488 nm/500-530 nm; PI 555 nm/591-635 nm.
To image the ProTCSn::ntdTomato, ProDR5v2::n3GFP dual-marker line, seedlings were fixed in 4% paraformaldehyde and treated with ClearSee solution (Kurihara et al., 2015 (link)) for five days. The seedlings were then stained with 0.1% (v/v) calcofluor white 2MR (Sigma-Aldrich, USA) in ClearSee solution for 30 min and observed under a confocal microscope (Leica TCS SP8) with preset excitation/emission wavelengths of 488 nm/500-530 nm for GFP, 550 nm/580 nm for tdTomato and 350 nm/420 nm for calcofluor white 2MR. To visualize the GFP protein in other transgenic lines, seedlings were stained in a 1 μM propidium iodide (PI; Sigma-Aldrich, USA) solution for 2 min and observed under a Carl Zeiss LSM700 confocal microscope (Carl Zeiss, Germany) with the following excitation and detection windows: GFP 488 nm/500-530 nm; PI 555 nm/591-635 nm.
