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The DAB and NBT staining

GN Guojie Nai
GL Guoping Liang
WM Weifeng Ma
SL Shixiong Lu
YL Yanmei Li
HG Huimin Gou
LG Lili Guo
BC Baihong Chen
JM Juan Mao
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3, 3, 9-Diaminobenzidine (DAB) and Nitrotetrazolium Blue Chloride (NBT) staining methods were performed as previously described [76, 77]. 10 mM 2-(N-Morpholino) ethanesulfonic acid (MES) was dissolved in distilled water and PH was 5.5. 1 mg/L DAB (Sigma-Aldrich) was dissolved in MES Buffer Solution and PH was 5.5. 0.5 mg/L NBT (Sigma-Aldrich) was dissolved in distilled water and PH was 5.8. Prepared DAB and NBT stain solution were stored at 4 ℃ and kept in a dark place. The destaining solution was ethanol: lactic acid: glycerin (3:1:1). Detached Leaves of tomatoes were immersed in culture dishes containing DAB and NBT staining solution for 12 h. The stained leaves were boiled with 95% ethanol for 10 min and leaves were decolorized with a destaining solution for 2 h. Decolorized leaves were placed in culture dishes with distilled water and photoed in EPSON Scan of root scanner (Regent company, Canada).

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