HISTOCHEMICAL DIFFERENTIATION OF ANTERIOR

COUPLING AND MUCOPROTEIN STAINS PITUITARY CELL TYPES BY CONTRASTING AZO-

BENJAMIN H. LANDING and HAZEL E. HALL, Departments of Pathology and Pediatrics of T h e Children’s Hospital and

Children’s Hospital Research Foundation, and the University of Cincinnati College of Medicine, Cincinnati, Ohio

Received for publication March 5, 1956

ABSTRACT.-AZO compounds couple with aromatic amino-acid nuclei in the cytoplasmic proteins of human anterior pituitary acidophile and basophile cells. In acidophile cells the reaction ap- pears to be due primarily to tyrosine, but also in part to histidine. Procedures are given for the use of naphthanil diazo blue B (tetrazo- tized di-ortho-anisidine), with or without a second coupling agent (H acid; S-amino-l-naphthol-3,6-disulfonic acid), to demonstrate acidophile cells, in contrast to mucoprotein stains (periodic acid oxidation followed by leukofuchsin or leukothionin) for the cyto- plasm of basophile cells. Evans blue can also be used to give a contrasting color to basophile cells.

In the course of attempts to determine the chemical nature of the acid-dye-binding substance of the cytoplasm of human anterior pituitary acidophile cells, a number of variations of the coupled tetrazonium reaction have been observed to provide histologic dif- ferentiation between these and the basophile cells. These studies were done in an effort to determine whether protein-bound histidine is respgnsible for the acidophilia, but the strong coupled tetrazoniurn reaction of acidophile cell cytoplasm is apparently due largely to a high content of protein-bound tyrosine. Such cells give a strong Millon reaction (Pearse, 1953, p. 404) for tyrosine, a negative Serra- Lopez reaction (Serra, 1946) for tryptophane, and a weak iodination- tetrazonium reaction (Landing and Hall, 1956) for histidine. The coupled tetrazonium reaction of the acidophilic cells is not affected by prior treatment with performic acid, as should be the case if tryptophane were responsible (Pearse, p. 56), but is abolished by benzoylation (Pearse, p. 415) and distinctly weakened by dinitro- fluorobenzene (Pearse, p. 41 6), results consistent with the interpreta- tion that the reaction is due primarily to tyrosine, but also in part to histidine.

Because the coupled tetrazonium reaction described by Pearse lThis study was supported by Research Grant #C-2143C, from the National

STAIN TECHNOLOGY, VOL. 31, No. 5, SFMFMEER 1956

Cancer Institute of the National Institutes of Health, USPHS.

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191 STAIN TECHNOLOGY

(p. 415) was tound to work well on human anterior pituitaries fixed in neutral 10% formalin and embedded in paraffin, tissue prepared in this way was used throughout the study. Naphthanil diazo blue B (tetrazotized di-ortho-anisidine)' has been utilized throughout as the first coupling reagent. It gives a yellow-brown color to sites of aromatic nuclei capable of coupling with azo groups. For a simple coupled tetrazonium reaction, H acid (8-amino-I-naphthol-3, 6- disulfonic acid)3 is satisfactory as a second coupling agent, and pro- duces a moderately dark brown to purple brown color on combina- tion with diazo blue B. By these methods, however, both acidophile and basophile cells react; although the former tend to stain some- what darker, distinction between the two cell types is not adequate for histologic use. Satisfactory histologic differentiation of the two cell types can be obtained by counterstaining the basophile cells. Of many dyes tested for this purpose, the one which provides the best color contrast to diazo blue B alone appears to be Evans blue (1% aqueous solution at pH 10 for 1 hr following treatment with diazo blue B).

More desirable from the histochemical point of view, however, are procedures for which the chemical nature oE the substrates bind- ing the various dyes can be stated. In an effort to develop such pro- cedures, using the azo-coupling property of acidophile cells as one aspect, attention was turned to the use of stains for mucoproteins, since the gonadotropic hormones and the thyrotropic hormone of basophile cells are mucoproteins (Ladman and Barrnett, 1955). The periodic acid-leukofuchsin procedure for mucoproteins does not give a color which contrasts well with that of the diazo blue B or with that of diazo blue B coupled to H acid. However, the leuko- thionin reagent of Van Duijn (1956) does give such contrast. Direc- tions for a coupled tetrazonium periodic acid-leukothionin sequence are given below; by this method, basophile cells are stained blue and acidophile cells brown to red-brown. Another variation of the azo-coupling method, depending on the enhanced affinity of acido- phile cell cytoplasm for certain dyes after treatment with diazo blue B, is also possible. Fast green FCF and light green SF yellowish both stain acidophile cells much more strongly after diazo blue B than without it. When this sequence is used in conjunction with the periodic acid-leukofuchsin reagent for mucoproteins, basophile cells are stained dark red to red-black, and acidophile cells bright green. Directions for this procedure are also given below.

*Obtained from Dajac Laboratories, Leominster, Mass. Note that the names used in the dye industry for diazonium compounds refer to the color obtained 011

coupling them to certain aromatic nuclei, not to the color of the compound ibelf. Thus, naphthanil diazo blue B is light brown rather than hlue, diazo black I( salt is red, etc.

30btained from Fisher Scientific Cumpan), Pitthurgh, Pa.

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AZO-COUPLING AND MUCOPROTEIN ST~4IN.S 195

The substance or substances, presumably protein in nature, re- sponsible for the azo-coupling reaction of anterior pituitary acido- phile and basophile cells has not been identified. Prolactin and growth hormone, made by the acidophile cells, contain tyrosine, and histidine, as also does the follicle-stimulating hormone of the baso- phile cells. However, whether the entities being demonstrated in the procedures given can be equated with these or other pituitary hor- mones remains to be established.

PROCEDURES

A. Coupled tetrazonium-leukothionin.

buffer, pH 9.2.

veronal-acetate buffer, pH 9.2) for 15 min at 0" C.

1. Take paraffin sections to water and rinse in veronal-acetate

2. Treat in naphthanil diazo blue B solution (50 mg in 50 ml of

3. Rinse in buffer solution. 4. Treat in H acid solution (2% in veronal-acetate buffer, pH 9.2)

5. Wash in running water for 2 min. 6. Oxidize in periodic acid for 10 min at room temperature, (I

gm of sodium periodate and 0.5 ml of nitric acid in 100 ml of distilled water).

for 15-30 sec.

7. Wash in running water for 5 min. 8. Stain in leukothionin solution for 2 hr. 9. Dehydrate with ethanol, clear in xylene, and mount.

Results: basophile cells, blue; acidophile cells, brown; chromophobe cells, unstained.

The leukothionin reagent (Van Duijn) is prepared as follows: Add 500 mg of thionin to 250 ml of water, boil for 5 min, cool to room temperature, and add distilled water to restore the original volume. Add 250 ml of tertiary butyl alcohol, 75 ml of 1 N HCI, and 5 gm of sodium bisulfite. Let stand for 24 hr at room temperature and then 48 hr at 0" C. Filter the amount necessary for each staining procedure, and return it to the stock bottle after use. The reagent keeps for some time in the refrigerator.

B. Naphthanil diazo blue B - a c i d green-periodic acid leukofztchsin procedure.

1. Take paraffin sections to water, and rinse in veronal-acetate buffer, pH 9.2.

2. Treat in naphthanil diazo bIue B soIution (50 mg in 50 ml of veronal-acetate buffer, pH 9.2) for 15 min at 0" C.

3. Rinse in buffer solution and then in water. 4. Stain in 3% aqueous fast green FCF or light green SF yellowish

for 15 min.

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196 STAIN TECHNOLOGY

5. Wash in running water for 2 min. 6. Treat in 5% aqueous phosphotungstic acid for 3 min. This

7. Wash in running water for 3-5 min. 8. Oxidize in periodic acid for 15 min at room temperature ( 1 gm

of sodium periodate and 0.5 ml of nitric acid in 100 ml of distilled water).

step prevents loss of the green dye during subsequent steps.

9. Wash in running water for 5 min. 10. Treat in leukofuchsin (bisulfite formula) for 20 min. 1 1 . Wash in running water for 10 min. 12. Dehydrate with ethanol, clear in xylene, and mount.

Results: basophile cells, dark red to red-black; acidophile cells, bright green; chromophobe cells, unstained or pale green.

REFERENCES LADMAN, A. J., and BARRNEIT, R. J. 1955. Localization of glycoprotein hor-

mones of the adenohypophysis by combined histochemical, physiochemical, and bioassay procedures. J. Clin. Endocrinol. Metab., 15, 871.

LANDING, B. H., and HALL, H. E. 1956. Selective demonstration of histidine. Stain Techn., 31, 197-200. [Paper 2, to follow this one]

PURSE, A. G. E. Histochemistry, Theoretical and Applied, pp. 56, 414, 415, 416. Little, Brown and Co., Boston.

SERRA, J. A. Histochemical tests for proteins and amino acids; the char- acterization of basic proteins. Stain Techn., 21, 5-18.

VAN DUIJN, P. 1956. A histochemically specific thionin-SO, reagent and its use in a bicolor method for deoxyribonucleic acid and periodic acid Schiff posi- tive substances. J. Histochem. Cytochem., 4, 55-63,

1953.

1946.

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