TECHNICAL NOTE |
Correspondence to: Guy Guidry, Neural Development Section, NINDS, NIH, Bldg. 36, Room 2B08, 36 Convent Drive, Bethesda MD 20892.
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Summary |
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A method is described for counterstaining tissue for use with the glyoxylic acid reaction for visual detection of biogenic amines. Counterstaining is achieved by addition of the fluorescent dye malachite green to the sucrosephosphateglyoxylic acid (SPG) solution used for processing of cryostat sections of unfixed tissues. When bound to tissues, the dye provides red-orange fluorescence of background tissue, which contrasts well with the green to yellow fluorescence induced by the glyoxylic acid reaction product formed with biogenic amines. The counterstaining technique is demonstrated in a number of catecholamine-containing peripheral tissues and is compared to sections that were processed without counterstaining. (J Histochem Cytochem 47:261264, 1999)
Key Words: biogenic amines, catecholamine, histofluorescence, glyoxylic acid, counterstain, malachite green
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Introduction |
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The glyoxylic acid condensation reaction is commonly used for visual detection of biogenic amines in histological sections. This method is a refinement of a family of techniques for fluorescence histochemistry of monoamines, all based on the cyclization of ethylamine side chains by highly reactive aldehydes, either formaldehyde or glyoxylic acid, producing compounds which fluoresce under suitable illumination (
The observation that many commonly used histological dyes are fluorescent suggested that it should be possible to add a fluorescent stain to the glyoxylic acid reaction solution to counterstain background tissues that are not normally visible with the SPG technique. Investigation of a number of dyes in that respect, including rose benzal, brilliant green, and light green sf yellowish, indicated that the cationic dye malachite green provides excellent contrast with monoamine histofluorescence. Under the excitation wavelengths used for catecholamine histofluorescence, the dye emits orange to red light which complements the aldehyde-induced blue-green fluorescence of catecholamine-containing cells and processes, particularly useful for tissue samples prepared for photomicrography.
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Materials and Methods |
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Adult and postnatal Day 10 (P10) SpragueDawley rats (Taconic; Germntown NY) were sacrificed with ether gas. Tissues were dissected, collected on ice, frozen in embedding medium (TBS Tissue Freezing Medium; EMS, Ft Washington, PA), and sectioned in a cryostat. All procedures involving the use of animals were in compliance with National Institutes of Health Animal Research Advisory Committee guidelines for intramural research.
Two solutions are required. The stock SPG solution (
Twelve-µm cryostat sections of frozen, unfixed tissue were melted onto uncoated glass slides and dipped immediately in the glyoxylic acid/malachite green solution for 5 sec. The slides were then transferred immediately to a Coplin jar containing glyoxylic solution without malachite green for 5 sec while stirring the solution with the slide to remove excess dye. The slides were removed and excess solution was wiped from areas around the tissue section and the back of the slide. As in the original report of
In some instances this method may produce sections that are overstained with malachite green. For example, sections of developing rodent footpad typically bind more dye than the other tissues that we examined. This overstaining tends to wash out the glyoxylic acid-induced catecholaminergic histofluorescence. Reducing the concentration of malachite green in the SPG solution to 0.001% and excluding the SPG wash step in the protocol produces sections that are less brightly counterstained and in which catecholamine histofluorescent axons appear brighter in sections of rodent footpad (
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Results and Discussion |
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A variety of catecholamine-containing peripheral tissues were processed with the SPG/malachite green solution and compared to sections that lacked the counterstain (Figure 1). In sections of adrenal gland, catecholamine-secreting medullary cells (arrows, Figure 1A) were typically brightly fluorescent with the SPG method. With the addition of malachite green (Figure 1B), regions of the adrenal medulla lacking catecholamines and the entire adrenal cortex become visible. Catecholaminergic axons in the small intestine (arrows, Figure 1C) are clearly localized in the muscle layers near the outer edge of the gut wall in counterstained sections (Figure 1D). In sections of kidney stained with SPG (Figure 1E), sharp, brightly fluorescent axons are seen in sections of the cortex containing convoluted tubules, which were faintly visible. With counterstain (Figure 1F), the convoluted tubules and as catecholaminergic axons are evident. In the spleen, sympathetic axons innervating splenic vasculature are histofluorescent after SPG treatment (Figure 1G), and in counterstained sections (Figure 1H) additional structures in the section are apparent. Catecholaminergic innervation of vessels is clearly visible within the centers of densely counterstained white pulp, separated by regions of red pulp that are less densely counterstained. In rat hairy skin, pilomotor fibers innervating piloerrectors (arrows, Figure 1I) are clearly associated with the base of hair shafts in sections stained with malachite green (arrows, Figure 1J). In sections of P10 rat footpad, catecholaminergic sympathetic axons innervating the pad, which are visible in SPG stained sections (arrows, Figure 1K and Figure 1L) are evident in association with developing sweat glands (asterisks) in counterstained sections.
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The mechanism of malachite green staining in aqueous solution is not readily apparent. In some instances, e.g., in convoluted tubules of the kidney cortex, the stain appears to be concentrated in cell nuclei. In other tissues, such as spleen and footpad, staining is more diffuse. Although the specific substrate to which the dye adheres is not clear, candidates include fatty acids, fatty aldehydes, phospholipids, glycolipids, and cholesterol, all of which are stained by the dye on thin-layer chromatographs (
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Literature Cited |
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