Plant Superoxide Anion Stain (NBT)

Plant tissues produce a variety of reactive oxygen species (ROS) under stress conditions. ROS activity is very large and extremely unstable, so the detection of ROS usually depends on its final products. Superoxide anion is a kind of reactive oxygen species, belonging to a kind of oxygen-containing free radical, can reduce NBT (nitro-blue tetrazolium) to water insoluble blue formazan compound, thereby locating superoxide anion in tissues. It is also known as the NBT method based on basic principles. This product is based on the above principle for superoxide anion staining in plant living tissue. It is generally applied to the whole staining of tender root tips, leaves, etc. After staining, the site of superoxide anion accumulation is blue to dark blue.

Wet ice transportation; Store at 2-8℃ away from light, valid for 12 months.

1. Reagent preparation: 50 mg NBT was fully dissolved in 100 mL Tris buffer (pH 7.4) to obtain NBT staining working solution, which was stored at 4℃ under light and effective within one week.

2. Collect seedlings or root tips, wash them with pure water, and blot the excess water on filter paper. Immerse the plant seedlings or root tips in NBT staining working solution at room temperature and avoid light for 2-6 h until the positive parts appear dark blue, and the rest parts are light blue or nearly colorless or the color of the plant itself (according to the degree of youth of the plant, the degree of color development adjusts the dyeing time).

3. Carefully remove plant seedlings or leaves with tweezers, rinse with pure water for 3-5 times, put them on filter paper to blot out excess water, and then immerse them in 95% ethanol for 3-16 h at 40℃ to remove the chlorophyll or light blue background of plant seedlings or leaves. Fresh 95% ethanol can be replaced many times during treatment.

4. Take out seedlings or leaves with tweezers, dip them into pure water and rinse them 3-5 times, put them on filter paper to drain the excess water, transfer the samples into an appropriate amount of NBT sample preservation solution and soak for 30 min, then take out the photos. Samples can be stored in the solution at room temperature for one week.

1. After preparing the NBT staining solution, it should be stored at 4℃ in the dark and used within a week. Storage time is too long, will affect the color.

2. Since any external factors may stimulate plant stress to produce superoxide anion, plant samples should be collected fresh and stained as soon as possible. Negative and positive blank control group is recommended.

3. Take photos as soon as possible to save the results after sample staining.

4. Wear a lab coat and disposable gloves during operation.

For Research Use Only!