Plant tissues produce a variety of reactive oxygen species (ROS) under stress environmental conditions. ROS activity is very large and extremely unstable, so the detection of ROS is usually determined by its end products.Superoxide anion is a kind of reactive oxygen species, which is an oxygen-containing free radical. It can reduce NBT (nitrogen blue tetrazole) to the insoluble blue formazan compound, thus locating the superoxide anion in tissues.It is also called the NBT method based on basic principles.Based on the above principles, this product is used for superoxide anion dyeing in plant living tissue.It is generally applied to the whole dyeing of tender root tips and leaves. After dyeing, the sites where superoxide anions gather appear blue to dark blue.
Tris buffer solution（pH 7.4）
NBT sample solution
Plant superoxide anion stain (NBT)
1.Preparation of the reagent: 50 mg NBT was fully dissolved in Tris buffer solution (pH 7.4) of 100 mL to obtain the NBT dyeing solution. The solution was stored at 4℃ away from light and effective for one week
2.Collect plant seedlings or root tips. Rinse with pure water and blot excess water on filter paper.The plant seedlings or root tips were immersed in the NBT dyeing solution, and the positive parts appeared dark blue, and the other parts were light blue or nearly colorless or the color of the plant itself (according to the tender degree of the plant).Color rendering level Adjust dyeing time)
3.Carefully remove the plant seedlings or leaves with tweezers, rinse them with pure water for 3-5 times, put them on filter paper and blot the excess water, then immerse them in 95% ethanol for 3-16 h at 40℃, in order to remove the chlorophyll or light blue background of the plant seedlings or leaves. Fresh 95% ethanol can be replaced several times during the treatment.
4.Remove plant seedlings or leaves with tweezers, immerse them in pure water and rinse them back and forth for 3-5 times. Place them on filter paper and blot the excess water. Transfer the samples to an appropriate amount of NBT sample storage solution for 30 minutes, and then take photos.Samples can be stored in the solution at room temperature for one week.
1.After preparation of the NBT dyeing solution, it should be stored at 4℃ away from light and used within one week.Storage time is too long, will affect the color.
2.Because any external factors may stimulate plant stress to produce superoxide anions, plant samples should be collected fresh and stained as soon as possible.Negative and positive blank control groups are recommended.
3.Take photos of the samples as soon as possible after staining.
4.Please wear a lab coat and disposable gloves when operating.
This product is only for scientific research purposes, not for clinical diagnosis.
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