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.

actin buffer (100mM PIPES, 10mM EGTA, 5mM MgSO4 and 0·3 M mannitol,pH 6·9), containing 2% (w/v) glycerol (1,2,3-propane-triol,99% pure, UCB) and 0·198 mM RLP (rhodamine-labelled phalloidin, Molecular Probes, stock dissolved in methanol).To avoid plasmolysis, the glycerol concentration has to be adapted to the cell type used. For staining the tobacco cells it was brought to 1% (w/v). Actin labelling was observed after 35 min and was further evaluated with time.


OLYSLAEGERS, Improved staining of F-actin and co-localization of mitochondria in plant cells. Journal of Microscopy, Vol. 192, Pt 1, October 1998, pp. 73–77., 1998.