Histochemical detection of H2O2 and O2- in rice leaves

Detection of hydrogen peroxide (H₂O₂) was done by 3, 3-diaminobenzidine (DAB) staining [42] and of superoxide radical (O₂^-) by NBT staining [43] in leaf segments of both stressed (Al conc. 25, 50, 100μM) and unstressed rice leaf segments. The leaf segments were immersed and infiltrated under vacuum with 1.25 mg/mL DAB staining solution, pH 7.8, dissolved in H₂O for 6 h, and 3 mg/mL nitro-blue tetrazolium (NBT) staining solution in 10 mM potassium phosphate buffer (pH 7.0) staining solution containing 10 mM NaN₃ for 30 min at room temperature. Stained leaves were bleached in acetic acid:glycerol:ethanol (1:1:3 v/v) solution at 100°C for 5 min, and stored in glycerol:ethanol (1:4 v/v) solution until photographed.