Shanklin, W.M. and Nassar, T.K.: Acta anat. 36: 257-264 (1959)

Department of Histology, School of Medicine, American University of Beirut, Lebanon

A PRELIMINARY SURVEY OF SOME
CYTOPLASMIC CONSTITUENTS STAINED BY
LUXOL FAST BLUE

By WILLIAM M. SHANKLIN and TAMIR K. NASSAR

Introduction


Kluver [1944] found that the fluorescent spectrum of the white matter of the central nervous system revealed a well-defined emission band at 630-620 mµ in 25 species of mammals and birds examined. Kluver states that this band strongly suggests a porphyrin. This earlier discovery led Kluver and Barrera [1953] to explore the possibility of staining myelin sheaths by means of porphyrin derivatives. They found one of these derivatives copper phthalocyanine particularly satisfactory. They discovered that by far the best myelin sheath preparations could be obtained by using Luxol Fist Blue MBS (DuPont), which is an alcohol-soluble amine salt of sulfonated copper phthalocyanine.

Pearse [1955] used the copper phthalocyanine as phospholipid stain. He used Methasol Fast Blue 2 G (I.C.I. Ltd.) the nearest British equivalent of Luxol Fast Blue. Margolis and Pickett [1956] used Luxol Fast Blue MBS (DuPont) in combination with other dyes to stain the myelin sheaths and other constituents of the nervous system.

It was our successful application of Luxol Fast Blue to myelinated fibres in the central nervous system that led us to try this stain on other tissues.

It is the purpose of this paper to describe briefly some of the cytoplasmic constituents of the small intestine, stomach, salivary glands, liver, pancreas, adrenal and brain as revealed by Luxol Fast Blue.

Material and Methods

  1. Fresh tissues from the albino rat were fixed by the dichromate-subli mate method (Elftman [1957]) for 3 days at room temperature.
  2. Tissues were washed over night in tapwater, dehydrated, cleared, embedded in paraffin and sectioned at 5µ. They were deparaffinized, hydrated through alcohols and placed in Gram's iodine to remove the mercury, followed by 5% hypo.
  3. Sections were passed through 3 changes of 95% arcohol and transferred direct to Luxol Fast Blue warmed in an incubator at 55°C from half to one hour. Excess stain was removed with 95% alcohol followed by distilled water.
  4. Differentiation was started by quickly immersing the sections in a 0.05% aqueous solution of lithium carbonate for 3 to 5 seconds, followed by 4 changes of 70% alcohol. Considerable care was exercised in obtaining the right degree of differentiation as this is a very delicate process. The final alcohol must be kept free of the stain. Sections were washed in distilled water and checked with the microscope. In case further differentiation was necessary the step was repeated quickly.
  5. Tissues were washed in water and counterstained with a 1.0% aqueous solution of erythrosine for 5-20 seconds. They were then washed in tapwater, dehydrated, cleared in xylol and mounted.

Luxol Fast Blue Solution

Luxol Fast Blue MBS - Microme Brand, Edward Gurr, London, was used in these studies. One grain of the dye was dissolved in 1000 cm3 of 95% alcohol. To this was added 5 cm3 of 10% acetic arid. This solution was filtered before use. The solution is very stable and keeps for some months.

Observations

Small Intestine. Epithelium lining the villi contained abundant closely packed granules stained dark blue by the Luxol Fast Blue (fig. 1). These granules filled most of the cytoplasm both above and below the nuclei. No positively stained material was observed in the striated border. Mucous in the goblet cells was not stained by Luxol Fast Blue. In passing from the upper part of the villus toward the base there was a progressive decrease in the blue stained granules. These granules were sparse in the epithelium of the intestinal glands. An exception were the cells of Paneth. The large granules in the cells of Paneth were stained dark blue.

Stomach. The surface lining and mucous neck cells were stained reddish and were negative to Luxol Fast Blue. The chief or zymogenic cells were also mostly negative. In contrast the parietal cells were filled with intensely stained deep blue granules.

  

Plate 1. Explanation of figures. All are photomicrographs of sections stained by Luxol Fast Blue counterstained with erythrosine. All magnifications are x 850. - Fig. 1. Section through a villus, apical end is toward the right. Note the sharp decrease in granular material in the epithelium toward the base of the villus. - Fig. 2. Section through salivary gland. Note negatively stained acinar cells, but deeply stained granules in the duct epithelium. - Fig. 3. Section of liver showing abundant granular material in the liver epithelium. Lightly stained area lower left is the central vein.

  

Salivary Glands. In areas containing mucous glands the cytopasm of the acinar cells was Luxol Fast Blue negative, however the duct [?] in the area was filled with densely packed intensely blue stained granules (fig. 2). Most of the serous cells were also Luxol Fast Blue negative, but the epithelium even in the smaller intralobular ducts contained abundant dark blue granules.

Liver. Liver cells in all parts of the lobule were filled with large blue stained bodies clumped together (fig. 3). Although abundant in allparts of the lobule they were more numerous toward the central vein.Luxol Fast Blue stained granules were not seen in the cells lining the bile ducts nor in the calls of the sinusoids.

Pancreas. The acinar cells varied greatly in their staining with Luxol Fast Blue for some areas were fairly well stained while other areas were negative. Duct epithelium was not stained by Luxol Fast Blue. Most cells in the islands of Langerhans have intensely stained dark blue granular material. A few of the larger cells have relatively few positively stained granules.

Adrenal The glomerular zone had relatively few Luxol Fast Blue stained granules, but the fascicular zone was strongly positive (fig. 4). Many of the cells have intensely blue stained granules wedged in between the vacuoles. Other cells, devoid of vacuoles, have their eintire cytoplasm filled with densely packed darkly stained granules. Cells in the the reticular zone varied greatly in the amount of Luxol Fast Blue material prcsct. No Luxol Fast Blue material was present in the medullary cells. Due to the intensely stained granules in the reticular zone, and their complete absence in the medulla there is a sharp line of demarkation between cortex and medulla (fig. 5).

Kidney. On the basis of differences in intensity of staining by Luxul Fast Blue the kidney is easily divided into cortex and medulla, furthermore the medulla is divided into an outer and an inner zone. The cortex was intensely stained (fig. 6), the outer medullary zone was moderately stained, but the inner medullary zone was devoid of positively stained granules. In the cortex the epithelium of the proximal, ascending and distal convoluted tubules contain intensely stained granular material. These granules are round or short rod shaped bodies occupying the basal two thirds of the cell in the proximal convoluted tubules. The abundant granules in the ascending limb and distal convoluted tubules are smaller. In some places irregular areas oof dark blue stained more homogencous materiaø is present in the cortical tubules. That part of the cell lying above the level oof the nuclei, also the brush border is devoid of blue stained granules. A small amount of positively stained material is seen in some glomeruli. The basement membranes around the proximal convoluted tubules were stained deep blue. Epithelial cells lining the lesser calyx have no blue stained granules.

Brain. Myelinated fibres were stained deep blue. In the human brain the linofuscin granules were also stained dark blue by the Luxol Fast Blue.

  

Plate 2. Explanation of figures. - Fig. 4. Section through the fascicular zone of the adrenal. Fig. 5. Section of adrenal including junction of darkly stained reticular zone with the unstained medulla. - Fig. 6. Section through cortex of kidney showing darkly stained granules in the kidney tubules.

  

Discussion

No attempt was made in this preliminary study to include all tissues and organs. In the small intestine numerous Luxol Fast Blue stained granules were observed in the lining cells of the villi and in the cells of Paneth. Parietal cells in the stomach were intensely stained. Neither mucous nor serous cells in the salivary glands were stained, however the duct epithelium contained strongly positive granules. It is interesting to note that the duct epithelium of the pancreas was not stained. Darkly stained clumps of granules were a constant finding in the liver cells. Most cells in the island of Langerhans had some deeply stained granules. The fascicular zone of the adrenal contained very abundant Luxol Fast Blue stained granules, but the medullary cells were completely negative. Tubules in the cortex and outer medullary zone of the kidney had positively stained granules. These were seen as darkly stained discrete rod-shaped granules in the proximal convoluted tubule and as smaller more diffuse granules in the ascending limb and distal convoluted tubule. In general glomeruli and the collecting duct system had no blue stained granules. The myelin sheaths in the brain of the rat and man were intensely stained. In the human brain the lipofuscin was also stained dark blue.

It should be emphasized that in all cases above where Luxol Fast Blue positively stained granules were described, these were in precisely those areas having the greatest physiological activity. The dichromate-sublimate method was used by Elftman [1957] for mitochondria and phospholipids in general. We have not determined, at this point, the nature of our cytophasmic constituents.

Kluver [1944], as well as Kluver and Barrera [1953] attributed the staining of the myelin sheath to the presence of a porphyrin. Our studies throw no further light on the chemical nature of the material stained. We believe, however, that the diverse granules stained in widely differing organs have in common some chemical constituent which is responsible for their staining reaction with Luxol Fast Blue.

Pearse [1955] attributes the staining of myelin sheaths by the copper phthalocyanins to the presence of phospholipids. We agree with Pearse that the copper phthalocyanins as dyes have considerable merit for they are easy to apply and they produce a brightly coloured, stable pigment.

Summary

Details are given of the technic used for staining organs by the Luxol Fast Blue method. This method stained granules deep blue in the epithelium lining the villi and cells of Panezh in the small intestine, the parietal cells of the stomach, the ducts of salivary glands, but not the duct epithelium of the pancreas, nor the epithelium of the bile ducts in the liver. Abundant darkly stained granules were observed in all liver cells. Most cells in the islands of Langerhans had small well stained granules. Abundant deeply stained granules were found in the kidney tubules, especially the proximal and distai.convoluted tubules and the ascending limb of Henle. Myelinated nerve fibres were stained deep blue.

Résumé

Les auteurs décrivent en detail une technique histlogique utilisant le Luxol Fast Blue. Ce produit colore en bleu foncé des granulations cytoplasmiques au niveau des cellules épithéliales du revêtemeni des villosités intestinales et des cellules de Paneth; des granulations identiques sont mises en évidence dans lea cellules bordantes de l'estomac et dans les épithélia des canaux des glandes salivaires. On ne les rencontre ni dans revêtement des canaux pancréatiques, ni dana l'épithélium des canaux biliaires. Des granulations nombreuses et fortement colorées se trouvent dans la cellule hépatique et dans l'épithélium des tubes rénaux, plus particulièrement dans les tubes contournés et la branche ascendante de l'anse de Henle. La plupart des cellules des ilôts de Langerhans renferment des granulations fines, bien colorées. Les fibres nerveuses à gaines de myéline sont colorées en bleufoncé.

Zusammenfassung

Einzelheiten der Technik einer Organfärhung mit Hilfe der Luxol-Blau. Methode werden beschrieben. Mit dieser Methode können Granula im Zottenepithel und in den Panethschen Zellen des Dünndarms, in den Wandzellen des Magens und der Ausführungsgänge von Speicheldrüsen tiefblau gefärbt werden, nicht jedoch im Epithel des Ausführungsgänge der Bauchspeicheldrüse und auch nicht un Gallengangspithel der Leber. Zahlreiche tiefblau gcfärbte Granula werden in allen Leberzellen gefunden. Die meisten Zellen der Langerhansschen Inseln enthielten kleine, gut gefärbte Körnchen. Zahlreiche tiefblaue Körnchen fanden sich in den Nierrenkanälchen, besonders in den proximalen und den distalen Tubuli contorti und dem aufsteigenden Schenkel der Henleschen Schleifa. Desgleichen wurden markhaltige Nervenfasern tiefblau gefärbt.

LITERATURE

Elftman, H.: Phospholipid fixation by dichromate-sublimate. Stain Technol. 32: 29 (1957).

Kluver, H.: On naturally occurring porphyrins in the central nervous system. Science 99: 482 (1944)

Kluver, H. and Barrera, E.: A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. exp. Neurol. 12: 400 (1953).

Margolis, G. and Pickett, J.P.: New application of the Luxol fast blue myelin stain.
I. A myelo-angio-cytoarchitectonic method; II. A myelin-neuroglia method; III. A myelin-fat method; IV. A myelin.axis cylinder method. Lab. Invest. 5: 459 (1956).

Copper phtnalocyanins as phospholipid stains. J. Path. Bact. 70: 554 (1955).


Received May 24th, 1958

The above article is transcribed from a copy at the AUB's Saab Medical Library.


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