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.Hydrogen peroxide is a type of reactive oxygen species.Under the catalysis of catalase, hydrogen peroxide can react with DAB (3, 3-diaminobenzidine tetrahydrochloride) quickly to produce browny-red compounds, thus locating hydrogen peroxide in tissues. According to the basic principle, it is also called the DAB method.Based on the above principles, this product is used for hydrogen peroxide dyeing in plant living tissues.It is generally applied to the whole dyeing of tender root tips and leaves. After dyeing, the sites where hydrogen peroxide accumulates appear brown to dark brown.
Phosphate buffer（pH 3.8）
DAB sample retention solution
Plant hydrogen peroxide staining liquid (DAB)
1. Reagent preparation: 100 mg DAB with phosphoric acid buffer 100 mL fully dissolved, to get DAB staining work liquid, to avoid light 4℃ preservation, effective within one week. Note: DAB is sensitive to light, the dissolution process needs to avoid light, if it is difficult to dissolve, ultrasound, magnetic stirring and other methods can be adopted for facilitating dissolution.
2. Collect plant seedlings or root tips under stress (such as heavy metal), wash slightly with pure water, and place on filter paper to absorb excess water. Plant seedlings or root tip immersed in DAB dyeing work dyeing liquid, at room temperature to avoid light staining 2-6 h, to the positive site appear dark brown, the rest of the site nearly colorless or the color of the plant itself can be. (Adjust the dyeing time according to the plant's tenderness and color rendering degree)
3. Remove the plant seedlings or leaves with tweezers, rinse them back and forth 3~5 times in pure water, place on the filter paper to absorb excess water, immerse in 95% ethanol at 40 degrees C to treat 3~16 h, with the aim of removing the plant seedlings or the leaf itself chlorophyll, during the decolorization period can be replaced with fresh 95% ethanol.
4. The plant seedlings or leaves with tweezers, rinse back and forth 3~5 times in pure water, put on the filter paper to absorb excess water, transfer the sample into the appropriate amount of DAB sample preservation fluid soaked 30 min, and then can be removed to take pictures. Samples can be kept at room temperature for one week in the preservation fluid.
1.DAB staining work liquid preparation after the need for 4 degrees C to avoid light preservation, within a week of use. Stored for too long, it affects color rendering.
2.Because hydrogen peroxide is easy to break down and any external factors can stimulate plant stress to produce hydrogen peroxide, plant samples need to be collected freshly and dyed as soon as possible. It is recommended to do a negative and positive blank control group.
3.Take a picture as soon as the sample dyeing is complete to save the results.
4.Wear lab clothes and disposable gloves when operating. Wear lab clothes and disposable gloves when operating.
This product is only for scientific research purposes, not for clinical diagnosis.
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