# **NBT stain**
Detect superoxide. Form insoluble dark-blue formazan precipitate after reduced by O2•−.
### Invitrogen ( N6495 ), Santa Cruz ( sc-296003A )
> Nitro blue Tetrazolium Chloride
## NBT working solution ( 1 mg / mL )
Dissolve the NBT powder in sodium phosphate buffer. The working solution must be protected from light.
| Chemical | M.W. ( g / mol ) | Concentration | Addition ( mg ) | Location |
| :---------- | :---: | :---: | :---: | :---: |
| NaH2PO4 | 119.98 | 1.342 mM | 161 | Ⅲ |
| Na2HPO4 | 141.96 | 8.657 mM | 1228 | Ⅲ |
| NaN3 | 65.01 | 10 mM | 650.1 | Ⅳ |
| NBT powder | 817.65 | — | 1000 | Ⅶ |
| ddH2O | | | 1 L | |
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- The working solution should be filtered before use. The quality among batches is not consistent. Some batches can be fully dissolved in the buffer, resulting clear yellowish working solution (the staining result is dark blue). However, some batches will form reddish brown precipitates ( the staining result will be reddish-darkbrown or greyish-darkblue, and tend to be over-stained ).
## **Procedure**
1. Directly immersed the root into the NBT solution ( pre-warmed to 30°C ), avoid the roots from touching the tube wall.
2. Vacumm 25 minutes under ~ 650 mmHg ( the lid should be opened ).
3. (optional) Close the lid and incubate under 30°C for 10 minutes ~ 2 hours, depend on the root condition.
- Generally, 10 mins is appropritate for young root ( less than one week old ), and the extremely old root ( more than two weeks old ) may require 2 hours.
4. Take picture under a dissection or light microscope.
Done !!!
## **Example image**
Figure 1. NBT staining in rice roots under different incubation conditions. Non-specific reddish staining occurs when using NBT solutions with excessive precipitate or when incubating at elevated temperatures (e.g., 37°C) (left). The optimized protocol yields characteristic, specific NBT staining (right).
