Whole seedlings were fixed in 1.5% (v/v) formaldehyde and 0.5% (v/v) glutaraldehyde made up in PEMT buffer(50 mm PIPES, 2 mm EGTA, 2 mm MgSO4, 0.05% [v/v] Triton X-100, pH 7.2) for 40 min and rinsed in PEMT buffer three times for 10 min. They were subsequently digested with 0.05% (w/v) Pectolyase Y-23 (Kikkoman) in PEM buffer (50 mm PIPES, 2 mm EGTA, 2 mm MgSO4) with 0.4m mannitol for 20 min, rinsed in PEM buffer three times,
treated with 220°C methanol for 10 min, and rehydrated in phosphate-buffered saline (PBS) for 10 min. Autofluorescence caused by free aldehydes from glutaraldehyde fixation
was reduced with 1 mg mL21 NaBH4 in PBS for 20min, followed by the treatment with 50 mm Gly in PBS(incubation buffer) for 30 min. Seedlings were incubated with primary antibodies against tubulin at room temperature overnight, rinsed in incubation buffer three times for 10 min, and secondary antibodies applied for 3 h at 37°C.After rinsing in PBS three times, root tips were cut off from the rest of seedlings, and mounted in 0.1% (w/v) paraphenylene diamine in 1:1 PBS-glycerol, pH 9. Cut cover glasses were used to space slide and cover glasses so as to avoid crushing delicate root tips.