4.2. 3,3-diaminobenzidine (DAB) Staining and Nitrioblue Tetrazolium (NBT) Staining

The 5-day-old seedlings were transferred to half strength 1/2 MS medium without or with 300 mM mannitol for 5 days, and then used for DAB or NBT staining to assay H₂O₂ or superoxide anion accumulation as we described previously [37,38,39]. For DAB staining, the seedlings were incubated in freshly prepared DAB staining solution (1 mg/mL DAB and 0.1% Tween 20 in 10 mM Na₂HPO₄) for 8 h, and then rinsed with 70% ethanol for several times to remove the chlorophyll. The images of the leaves were captured using a digital camera. For superoxide anion staining, the seedlings were vacuum infiltrated with 0.1 mg/mL NBT in 25 mM HEPES buffer (pH 7.6) for 2 h in darkness. Chlorophyll was removed by using 70% ethanol, and then the images of the leaves were captured using a digital camera. Three independent biological replicates were performed, and the relative intensity of DAB or NBT staining was quantitatively analyzed according to our previously reported method [37].