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 (Na2SO3) is applied to the specimen for 5-10 minutes. Lignin will stain orange to red.

4',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 0.010.05% (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.

Ferric chloride (FeCl3) is prepared as a 2% (w/v) aqueous solution, although for embedded tissues a 10% solution is recommended. The specimen is incubated in the solution for 5-10 min. Phenolic compounds, including tannins will stain yellow or orange with this reagent.

The Mäule reagent is used to distinguish between guaiacyl and syringyl residues in the lignin. A freshly prepared aqueous solution of 1% (w/v) potassium permanganate (KMnO4) is applied to the specimen for at least 30 min. After washing in distilled water for 2 min. and removing all liquid, a 15-20% aqueous solution of HCl is applied. The HCl is removed, and a 10% solution of NH3 is applied. Syringyl residues result in the development of a red color. Absence of syringyl residues results in the development of a yellow color. Chapple et al. (1992) used this reagent to demonstrate the lack of syringyl residues in the lignin of the Arabidopsis fah-1 mutant, in which the activity of the monolignol biosynthetic enzyme ferulate 5-hydroxylase (coniferaldehyde/coniferyl alcohol 5-hydroxylase) is reduced. Overexpression of a wild-type copy of the defective F5H gene in the fah1 mutant resulted in a predominance of syringyl residues, as evident from the red color of the lignified tissues in the stem (Meyer et al., 1998).

Methylene Blue is used to detect pectin and phenolic compounds. It is applied to the specimen as a 0.01-0.15% (w/v) aqueous solution, incubated for 30 min. at room temperature, or for 5 min. at 60°C, and then removed by washing in distilled water. Methylene Blue is a blue dye, but it produces a red coloration as a result of the reaction with the phenolic or pectic substances. This phenomenon is referred to as metachromasia, and the cell or tissue components that exhibit it are called metachromatic.

Toluidine Blue O is dissolved in a buffer with a pH between 4 and 8, or in 70% ethanol or in a 0.05% (w/v) borax solution and kept at 4°C. The specimen is stained for 1-10 min. washed for 1-2 min. in distilled water, or in the buffer used to dissolve the Toluidine Blue O, or in ethanol until all excess stain is removed. When the specimen is viewed under the microscope, different cell components produce colors different colors (metachromasia): DNA is bluish-green, RNA is violet, the middle lamella is red, non-lignified cell walls are red-violet or blue-violet, and polymerized phenolics such as lignin are green or blusih-green. This stain is therefore of general use.

Phloroglucinol-HCl is a 2% (w/v) solution of phloroglucinol (4.30) dissolved in a 2:1 mixture of ethanol and concentrated HCl. This reagent reacts with cinnamaldehyde end groups (4.31) in the lignin, resulting in the cationic chromophore (4.32), and which appears as a burgundy-red compound (Adler et al., 1948; Geiger and Fuggerer, 1979; Pomar et al., 2002; see Figure 4-6). This procedure is known in the literature as the Wiesner reaction and is also used as a general stain for lignin. This may not be appropriate if samples differing in cinnamaldehyde content are being compared. This is probably the most common stain used to detect lignin, because the reagent is easy to make and the reaction can be readily monitored. Pictures of the use of the Wiesner reaction can be found in Halpin et al. (1998), Vermerris et al. (2002), and Pomar et al. (2002).

Vanillin-HCl is prepared by making a 0.05% (w/v) solution in 50% ethanol. This is then mixed in a 2:5 ratio with concentrated HCl. The mix is applied to the specimen for 1 min. Alternatively, the sample is immersed in

Figure 4-6. Reaction of phloroglucinol with coniferaldehyde end-groups in the lignin under acidic conditions results in the formation of a red chromophore.

Figure 4-6. Reaction of phloroglucinol with coniferaldehyde end-groups in the lignin under acidic conditions results in the formation of a red chromophore.

a saturated solution of vanillin in 95% ethanol for 15-30 min. The section is then placed on a microscope slide, one drop of 9N HCl is added and the section is immediately viewed under the microscope. This stain will produce a red color in the presence of tannins. When pictures need to be taken, it is important to realized that this stain is not permanent.

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