Immunofluorescence Staining of Microtubules

We used 6-d-old seedlings (control) and 4-d-old seedlings treated with 2 μm ABA for 2 d for microtubule assays, as described in Deng et al. (2015). Root tips of 1-cm segments were cut and fixed in 4% (v/v) paraformaldehyde in PME buffer 1 (50 mm PIPES, 2 mm MgSO₄, and 2 mm EGTA [pH 6.9]) containing 0.05% (v/v) Triton X-100 for 30 min. After washing thoroughly with PME buffer 1, samples were digested with 2% (v/v) cellulase R-10 and 1% (v/v) pectolyase Y-23 (both from Yakult Pharmaceutical Industry) in PME buffer 1 at 37°C for 30 min to 1 h The softened root tips were washed gently with PME buffer 1 and frozen and thawed twice in liquid nitrogen. Samples were treated with the blocking buffer (3% [v/v] BSA in phosphate-buffered saline with Tween 20 [PBST; 137 mm NaCl, 2.7 mm KCl, 10 mm Na₂HPO₄, 1.76 mm KH₂PO₄, and 0.05% Triton X-100]) for 1 h at room temperature, then incubated with 1:50 diluted primary antibodies anti-α-tubulin (T9026, Sigma) in the blocking buffer at 4°C overnight. After washing with PBST five times, samples were incubated with 1:800-diluted secondary antibodies Alexa Fluor 488 goat antimouse IgG (A-11001, Thermo Fischer Scientific) in PBST at 37°C for 3 h. After washing four times with PBST and once with PBS, samples were mounted with PBS containing 50% (v/v) glycerol and 0.1% (v/v) o-phenylenediamine. Images were obtained with a 63× water objective (NA 1.2) under a Zeiss LSM 880 confocal laser scanning microscope. Fluorescence was excited at 488 nm and collected with a 492- to 560-nm filter. The overall fluorescence signal of each genotype was obtained under identical staining conditions and confocal settings 8% 488-nm laser power, 58-μm pinhole, and stack scanning mode.