Volume 34, No. 2, March 1959
Printed in U.S.A.


William M. Shanklin, Tamir K. Nassar and Marietta Issidorides

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

Received for publication July 16, 1958

Abstract.- Human pituitaries fixed in Bouin's fluid or 10% formalin were stained by the PAS, Masson trichrome and luxol fast blue methods. By comparing adjacent sections stained by these 3 methods it was found that the alpha cells which are PAS negative, but stained red in the Masson trichrome method, were intensely stained by luxol fast blue. The beta cells which are stained blue by the PAS and Masson methods were not stained by luxol fast blue. Similar observations were made on a series of pituitaries from 8 other mammalian species. It is concluded that luxol fast blue is a selective stain for alpha cells in the mammalian pituitary.

Luxol fast blue was used a a myelin sheath stain by Klüver (1944), Klüver and Barrera (1953), Margolis and Pickett (1956) and by Dziabis (1958). Klüver attributed this staining reaction to the presence of a porphyrin. Pearse (1955) found that methasol fast blue, the nearest British equivalent at that time to luxol fast blue, gave good results when used as a stain for phospholipids. We (Shanklin and Nassar, 1958) stained with luxol fast blue cytoplasmic constituents, whose identity was not determined, in the small intestine, stomach, salivary glands, kidney, liver, pancreas, adrenal and brain. German and Schwartz (1958) state that luxol fast blue is a selective stain for the beta cells of the pituitary.

It is generally accepted that the Masson trichrome method stains the pituitary alpha cells red and the beta cells blue, furthermore that periodic acid-Schiff staining (PAS) is positive for beta cells but negative for alpha cells.

It is our purpose to prove, by comparing sections of the human pituitary stained by the Masson and PAS methods with those stained by luxol fast blue, that luxol fast blue stains alpha cells an intense blue but does not stain the beta cells.


  1. Adult human pituitaries were fixed in Bouin's fluid or 10% formalin, embedded in paraffin, sectioned at 5µ, deparaffinized, and passed through absolute alcohol and 2 changes of 95% alcohol.
  2. Sections were transferred from alcohol direct to luxol fast blue and warmed in an incubator at 55°C from 2 to 4 hr. Excess stain was removed with 95% alcohol followed by distilled water.
  3. Differentiation was started by quickly immersing the sections in a 0,05% aqueous solution of lithium carbonate for 3 to 5 sec, 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 a microscope. In case further differentiation was necessary this step was repeated quickly.
  4. Tissues were washed in water and counterstained with a 1.0% aqueous solution of erythrosin for 5-10 sec. The were then washed in tapwater, dehydrated, cleared in xylene and mounted.

Luxol fast blue solution. Luxol fast blue MBS-Microme brand, Edward Gurr, London, purchased September 1957, was used in these studies. One gram of the dye was dissolved in 1000 ml of 95% alcohol. To this was added 5 ml of 10% acetic acid. This solution was filtered before use. The solution is very stable and keeps for some months.

Adult human pituitaries were also stained by the PAS method, as described by McManus (1948) for paraflin sections. and by the Masson trichrome method as modified by Foote (1933).

Adjacent sections of the pituitaries of cat, cow, dog, horse, jackal, pig, rabbit and rat were also stained by the PAS, Masson and luxol fast blue methods.


We present as specific evidence for our proposal sections from the pituitary of a 16-yr-old male Fig. 1, 2 and 3. We have used adjacent sections stained by the PAS, Masson trichrome, and luxol fast blue methods. Note the same area of colloid (C) and blood vessels in all three figures. In Fig. 1, from a section stained by PAS, there are no positively stained beta cells immediately surrounding the colloid, however there are areas of PAS stained beta cells (B) to the right side of the figure. In Fig. 2, from a section stained by the Masson trichrome method, cells immediately around the area of colloid (C) are light colored (A) and have no granules. On the slide these are stained bright red by the ponceau and acid fuchsin and are readily identified as alpha cells (A). To the right side in the figure are areas of darkly stained granular cells (B). On the sections these cells are stained dark blue and are identified as beta cells. On the basis of PAS and Masson-stained sections we conclude that alpha cells immediately surround the colloid and most of the adjacent area, while beta cells are limited mostly to the right hand side of the area.

In Fig. 3 from a section stained by luxol fast blue the cells (A) surrounding the colloid are darkly stained while those to the right (B) are more lightly stained. On the slide, the cells around the colloid are stained an intense blue color by luxol fast blue, while those to the right are stained light red by erythrosin. We conclude that. the luxol fast blue-stained cells correspond to the red-stained cells in Masson-stained sections and therefore are alpha cells. Furthermore the luxol fast blue-stained cells correspond to the PAS negative cells. In Contrast to this observation, the areas of PAS positively stained cells are not stained by luxol fast blue.

Fig. 1. Photomicrograph of a section of a human pituitary stained by the PAS method A, alpha cells; B, beta cells; C, colloid. Green filter. X 425.

Fig. 2. Photomicrograph of a section of a human pituitary stained by the Masson trichromc method. Red filter. x 425.

Pig. 3. Photomicrograph of a section of a human pituitary stained by the luxol fast blue method. Green filter. X 425.

The beta cells, following staining by the PAS or by the Mauon trichrome methods. were filled with fine granules. Granules were not observed in the luxol fast blue-stained alpha cells.

All three methods stained the colloid with varying degrees of intensity; very dark with PAS and Masson stains but relatively light by luxol fast blue.

A study of the pituitaries of cat, cow, dog, horse, jackal, pig, rabbit and rat showed that luxol fast blue is also a selective stain for the alpha cells in these animals.


Our studies demonstrate that luxol fast blue is a highly selective stain for alpha cells in the human and a series of other mammalian pituitaries. Our conclusion is diametrically opposed to the conclusion of German and Schwartz (1958).

Our findings throw no further light on the suggestion that the luxol fast blue staining reaction is due to the presence of a porphyrin (Klüver, 1944), or to phospholipids (Pearse, 1955).

The substance stained in the alpha cells was homogeneous, however, the material stained by luxol fast blue in our previous study (Shanklin and Nassar, 1958) in the epithelium of the intestinal villi, cells of Paneth, parietal cells of the stomach, ducts of the salivary glands, liver cells, islands of Langerhans, cortex of adrenal and kidney tubules was granular. The staining of the myelin sheath was also homogeneous.


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GERMAN, N. I., AND SCHWARTZ, H. J. 1958. A selective staining method for the beta-cells of the pituitary. Anat. Rec., 130, 410-11.

KLÜVER, H. 1944. On naturally occurring porphyrins in the central nervous system. Science, 99, 482-4.

KLÜVER, H.. AND BARRERA, E. 1953. A method for the combined staining of cells and fibers in the nervous system. J. Neuropath. Exp. Neurol., 12, 400-3.

MARGOLIS, C., AND PICKETT, J. P. 1956. New applications of the luxol fast blue myelin stain. I. A myeclo-angio-cytoarchitectonic method; II. A niyelin-neuroglia method: III. A niyclin-fat method; IV. A myelin-axis cylinder method. Lab. Investig., 5, 459-74.

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SHANKLIN, W. M. and NASSAR, TAMIR K. 1938. A preliminary survey of some cytoplasmic constituents stained by luxol fast blue. Acta Anat., in press.

1Research supported by a grant (B-1701) from the National Institutes of Health. United States Public Health Service.

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


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