Journal of Histochemistry and Cytochemistry, Vol. 47, 261-264, February 1999, Copyright © 1999, The Histochemical Society, Inc.


TECHNICAL NOTE

A Method for Counterstaining Tissues in Conjunction with the Glyoxylic Acid Condensation Reaction for Detection of Biogenic Amines

Guy Guidrya
a Neural Development Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland

Correspondence to: Guy Guidry, Neural Development Section, NINDS, NIH, Bldg. 36, Room 2B08, 36 Convent Drive, Bethesda MD 20892.


  Summary
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Summary
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Materials and Methods
Results and Discussion
Literature Cited

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 sucrose–phosphate–glyoxylic 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 47:261–264, 1999)

Key Words: biogenic amines, catecholamine, histofluorescence, glyoxylic acid, counterstain, malachite green


  Introduction
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Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

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 (Bjorklund 1983 ). Although catecholamine-containing cells and processes are brightly fluorescent with the sucrose–phosphate–glyoxylic acid (SPG) method (De la Torre 1980 ), the simplest and most rapid of the monoamine fluorescence techniques, surrounding tissue is usually not visible because of the lack of significant tissue autofluorescence. This can be particularly problematic in photomicrographs of fluorescent histological sections. One method for imaging unstained tissue, if the light emitted by fluorescing amines is relatively intense, is to combine low light level phase-contrast microscopy with reflected fluorescence.

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.


  Materials and Methods
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Materials and Methods
Results and Discussion
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Adult and postnatal Day 10 (P10) Sprague–Dawley 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 (De la Torre 1980 ) is made by dissolving 10.2 g sucrose, 4.8 g potassium phosphate monobasic, and 1.5 g glyoxylic acid monohydride (all from Sigma; St Louis, MO) in 100 ml water. The solution is brought to pH 7.4 with sodium hydroxide and then water is added to achieve a final volume of 150 ml. This solution is stable for several weeks at 4C. For the SPG/malachite green solution, 0.07 g malachite green (Eastman Kodak; Rochester, NY) was added to 40 ml SPG solution, vortexed vigorously, and then spun at 2000 x g for 30 min. The solution was transferred to a Coplin jar with a transfer pipette, taking care to avoid transferring any undissolved dye on the side and bottom of the centrifuge tube. The SPG/malachite green solution is usable for several hours at RT.

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 De la Torre 1980 , the sections were then dried under a strong stream of air from the benchtop air supply until the tissue was completely dried and turned slightly opaque. The tissue sections can be stored temporarily in a desiccated container through which dry air is circulated for batch processing, or can remain under the airstream. Additional drying for approximately 10 min is required for sharp catecholamine histofluorescence. The sections were subsequently covered with a drop of light mineral oil (Sigma) and heated to 95C for 2.5 min in a laboratory oven. The slides were removed, allowed to cool on the benchtop, and coverslipped with mineral oil. Sections were photographed using a Zeiss Axioplan 2 with appropriate filter cubes.

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 (Guidry and Landis 1998 ). Therefore, use of either the malachite green-saturated solution or 0.001% solution should be determined empirically for each type of tissue stained.


  Results and Discussion
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Materials and Methods
Results and Discussion
Literature Cited

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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Figure 1. (A) Catecholamine histofluorescent medullary cells (arrows) are visible in the core of the adrenal gland. (B) With the malachite green counterstain, the adrenal cortex is apparent. The catecholaminergic medullary cells maintain bright histofluorescence (arrow). (C,D) With counterstaining, catecholamine-containing fibers in the small intestine (C, arrow) are more easily localized in the muscularis externa region of the gut wall (D, arrow). (E) Catecholamine histofluorescent fibers (arrow) in adult rat kidney. (F) In counterstained sections of rat kidney, convoluted tubules (arrowheads) are evident surrounding catecholaminergic axons associated with a blood vessel. (G) Catecholaminergic sympathetic axons innervating rat spleen are indicated with an arrow. (H) In counterstained sections of rat spleen, catecholaminergic fibers (arrows) are visible within the splenic white pulp and in the splenic red pulp (arrowheads). (I) Pilomotor axons (arrows) in rat hairy skin and hair shafts (arrowheads) are visible. (J) In counterstained tissue, the surrounding tissue is evident. In this section, pilomotor fibers (arrows) arborized adjacent to a hair shaft base lacking fibers are visible (small arrowheads). In addition, dark collagen fibrils (small arrows) and shafts of hair are present. (K,L) Catecholaminergic axons (arrows) innervating P10 rat hind footpad in K, are clearly seen to associate with developing sweat glands (asterisks) in the counterstained section, 1. Bar: A = 0.2 mm for A, B, G, H; = 0.1 mm for C–F, I, K, L.

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 (Teichman et al. 1974 ). The absence of staining of glycerylphosphorylcholine and other phospholipid constituents under similar conditions suggests that staining may involve interaction with long-chain carbon groups (Teichman et al. 1974 ). The dye has also been employed as a staining reagent for prostaglandins on thin-layer chromatographic plates (Singh et al. 1975 ). In addition, malachite green is commonly used in conjunction with glutaraldehyde fixative in ultrastructural studies to retain lipid elements that are soluble in aqueous glutaraldehyde (Pourcho et al. 1978 ; Gould and Bernstein 1985 ).


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results and Discussion
Literature Cited

Björklund A (1983) Fluorescence histochemisty of biogenic amines. In Björklund A, Hökfelt T, eds. Handbook of Chemical Neuroanatomy. Vol 1. Methods in Chemical Neuroanatomy. Amsterdam, New York, Elsevier Science, 50-120

De la Torre JC (1980) An improved approach to histofluorescence using the SPG method for tissue monoamines. J Neurosci Methods 3:1-5[Medline]

Gould SF, Bernstein MH (1985) Effect of estrogen on the affinity of malachite green for staining cardiac lipid inclusions in mice. Acta Anat 123:25-29[Medline]

Guidry G, Landis SC (1998) Target-dependent development of the vesicular acetylcholine transporter in rodent sweat gland innervation. Dev Biol 199:175-184[Medline]

Pourcho RG, Bernstein MH, Gould SF (1978) Malachite green: applications in electron microscopy. Stain Technol 53:29-35[Medline]

Singh EF, Moawad A, Zuspan FP (1975) Malachite green—a new staining reagent for prostaglandins. J Chromatogr 105:194-196[Medline]

Teichman RJ, Takei GH, Cummins JM (1974) Detection of fatty acids, fatty aldehydes, phospholipids, glycolipids and cholesterol on thin-layer chromatograms stained with malachite green. J Chromatogr 88:425-427[Medline]