Plant Hydrogen Peroxide Staining Liquid (DAB)

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. Hydrogen peroxide is a type of reactive oxygen species. Catalyzed by catalase, hydrogen peroxide can rapidly react with DAB (3, 3-diaminobenzidine tetrahydrochloride) to form a brown-red compound, so as to locate hydrogen peroxide in tissues. According to the basic principle, it is also called DAB method. Based on the above principle, this product is used for hydrogen peroxide staining in plant living tissues. It is generally applied to the tender root tips, leaves and other overall staining, after staining the site of hydrogen peroxide accumulation is brown to dark brown.

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

1. Reagent preparation: 100 mg DAB was fully dissolved in 100 mL phosphate buffer to obtain DAB staining working solution, which was stored at 4℃ under light and effective within one week. Note: DAB is sensitive to light and needs to be shielded from light during the dissolution process. If it is difficult to dissolve, the dissolution can be promoted by ultrasonic and magnetic stirring.

2. Collect seedlings or root tips of plants under stress (such as heavy metals), wash them slightly with pure water, and put them on filter paper to drain the excess water. Plant seedlings or root tips were immersed in DAB staining working dye solution at room temperature and kept away from light for 2-6 h until the positive part appeared dark brown, and the rest parts were nearly colorless or showed the color of the plant itself. (Adjust the dyeing time according to the degree of youth and color development of plants)

3. Carefully remove the plant seedlings or leaves with tweezers, immerse them in pure water and rinse them back and forth 3-5 times, put them on filter paper to blot out the excess water, and then immerse them in 95% ethanol at 40℃ for 3-16 h. The purpose is to remove the chlorophyll of the plant seedlings or leaves themselves, and the fresh 95% ethanol can be replaced many times during decolorization.

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

1. After the DAB staining working solution is prepared, it should be stored at 4℃ and kept away from light, and used within one week. Storage time is too long, will affect the color.

2. Since hydrogen peroxide is easy to decompose and any external factors may stimulate plant stress to produce hydrogen peroxide, plant samples need to 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!