An overview of histochemical staining protocols

A number of histochemical stains are available to visualize phenolic compounds in the plant, either in thin sections, or applied to whole tissues. De Neergaard (1997) published a series of detailed protocols that provides an excellent source of information. Below follows a summary of the available reagents and the specific compounds they detect. 

Aniline sulfate is dissolved in 0.1N sulphuric acid in aqueous solution or 60% (v/v) ethanol to a final concentration of 1-6 % (w/v). This stain reacts with lignin, which will turn yellow. 

Chlorine sulfite can be used to detect syringyl lignin. The specimen is placed above a tissue soaked in a sodium hypochlorite ( bleach ) solution for 30 minutes. A 3% (w/v) solution of sodium sulfite (Na2S03) is applied to the specimen for 5-10 minutes. Lignin will stain orange to red. 

6 diamino 2-phenylindole (DAPI) is a fluorescent stain that reacts with DNA and phenolic compounds. It can be used on live tissue at a concentration of 0.002% (w/v) or in fixed tissue at a concentration of 0.01-0.05% (w/v). Several buffers, such as TRIS or potassium phosphate can be used, depending on the exact application the pH is generally around 7.5. The staining solution needs to be stored at 4°C. The specimens are incubated in the dark for a minimum of 30 minutes, up to an overnight incubation (4°C) and then exposed to UV light with an excitation wavelength of 365 nm. 

Ethidium bromide is a fluorescent dye that can be used for the visualization of DNA as well as lignin and other phenolic substances. An aqueous solution of 0.1 % (w/v) is prepared and applied to tissue sections for 5-10 minutes. The specimen is exposed to UV light, which results in an orange fluorescence as a result of the ethidium bromide. 

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