Okajimas Fol. anat. jap., 43: 291-297, 1967

Some Problems on the Leuco-Dye-Peroxide Reaction

The 31st Report of Histochemical Study of Peroxidase

by

Tadao Mitsui, Toshio Shinoda and Hiroshi Ogawa

Department of Anatomy, School of Medicine Keio University, Shinjuku, Tokyo

In general, for the histochemical demonstration of peroxidase, b

enzidine, naphthol, and leuco-dyes are utilized in the prosence of

hydrogen peroxide. Among these , the technical modifications of the benzidine-peroxide method are the most numerous . Alpha-naphthol has been used less often than benzidine . Application of the leuco-dye method for animal tissues is remarkably limited and this method

has been used chiefly for the demonstration of erythrocytes . The present study has dealt with the histochemical application

of the leuco-dye method and also with the comparison between

diamine-peroxide reaction and leuco-dye-peroxide reaction from the

standpoint of histochemical staining .

Materials and methods

For materials blood smears of horse , frozen sections of guinea pig skin and root stock of Japanese horse-radish were used . Eosino-phil leucocyte and neutrophil leucocyte are two of the richest sources of peroxidase in the blood tissue .

The fixatives for these materials were formol-alcohol (1 : 9) . The staining reagents were prepared as follows :

Benzidine-peroxide reagent : It contains 100 ml distilled water, 0

.3 g benzidine and 10 drops of 3 per cent hydrogen peroxide . Leuco-dye-peroxide reagent : To patent blue V solution (1 g in

100 ml distilled water) , or to phloxine (erythrosin BB) solution (1 g in 100 ml distilled water), and 10 g powdered zinc and 2 ml glacial

acetic acid. Boil until colorless . Cool and filter. Before use add to 10 ml of the stock leuco-dye solution one drop of 3 per cent hydrogen

peroxide.

* This work is dedicated to the memory of the late Professor Hiroshi Hosokawa ** Student of the School of Medicine , Keio University

291

292Tadao Mitsui, Toshio Shinoda and Hiroshi Ogawa

Staining time was generally five minutes. After washing

water, counterstain with hematoxylin or other adequate dyes.

Experimental Results

While carrying out an investigation into acid dyes that can b

utilized for the leuco-dye-peroxide reaction, it was found that phloxin

(erythrosin BB) can be used as substitute for patent blue. The materials which could be stained with the leuco-dye metho

were red blood cells, eosinophil granules of eosinophil leucocyte

and cell walls of the root stock of Japanese horse-radish. The fine ings obtained with the leuco-patent blue method are generally th

same as those obtained with the leuco-phloxine method . When a positive reaction occurs, the tissues or cells stain re

with the leuco-phloxine method. This red end-product is comparati

vely stable to various solvents as shown in Table 1.

Table 1. Decoloration test of stained tissue sections of horse-radish . + (not decolorized), (decolorized)

It should be noted that neutrophil leucocytes and monocyte

were not stained at all with the leuco-patent blue method nor wit

the leuco-phloxine method.

As is well known, neutrophil leucocytes and monocytes are we

stained with benzidine-peroxide solution, orthophenylenediamin(

peroxide solution or paraphenylenediamine-peroxide solution.

The eosinophil leucocytes of Triturus pyrrhogaster were nc

stained at all with the leuco-patent blue method showing the sam

results as those obtained with the benzidine-peroxide method. Fui

ther, the hair follicle cells of the guinea pig were not stained

all with leuco-patent blue-peroxide solution nor with leuco-phloxin(

peroxide solution, whereas these cells were distinctly stained with paraphenylenediamine-peroxide solution.

As shown in Table 2, leuco-patent blue-peroxide and leuco-

phloxine-peroxide reactions are not inhibited at all by methanol. For comparison, the results of benzidine-peroxide reaction are shown in the same Table, indicating that methanol is originally a powerful inhibitor of the ordinary peroxidase reaction of leucocytes.

The results of inhibition test with heating (100°C) are shown in Table 3. To our surprise, leuco-patent blue-peroxide reaction was not inhibited even by the treatment with 100°C for ten minutes. On the other hand, leuco-phloxine-peroxide reaction was inhibited by the treatment with 100°C for five minutes.

However, it should be noted that the eosinophil granules of eosinophil leucocytes treated with heating to 100°C for five minutes could not be stained with a simple aqueous solution of phloxine as

Table 3. Inhibition test of leuco-dye-peroxide reaction of horse eosinophil leucocytes with heating (boiling water).

+ (stained), — (not stained). Erythrocytes were stained in all cases except for

benzidine-Deroxide reaction.

294 Tadao Mitsui, Toshio Shinoda and Hiroshi Ogawa

shown in Table 3. This fact implies that, even though the red end-

product of leuco-phloxine-peroxide reaction occurs in these eosinophil leucocytes, this substance cannot be adsorbed to the eosinophil

granules due to the treatment with heating mentioned above. In the experiments of inhibition with alkaline solutions such

as potassium hydroxide (KOH) and potassium cyanide (KCN), it was made clear that the intensity of the leuco-dye-peroxide reaction of

erythrocytes was not changed, that the reaction of eosinophil leucocyte was generally inhibited, and that the leuco-phloxine-peroxide reaction showed a stronger resistance to these alkaline solutions than the ordinary benzidine-peroxide reaction (Table 4). Furthermore, it is

of interest that the leuco-phloxine-peroxide reactions of both eosino-

phil leucocytes and neutrophil leucocytes, were rather activated by one molar solution of potassium cyanide, though the cytoplasm of neutrophil leucocytes stained diffusely, and did not take a granular

pattern.

Table 4. Inhibition test of leuco-dye-peroxide reaction of horse eosinophil leucocytes with potassium hydroxide (KOH) and potassium cyanide (KCN). Blood specimens were exposed to KOH or KCN for 5 minutes after fixation with ethanol-formol for 1 minute. — (reaction was inhibited), + (slightly in-hibited), + (not inhibited), -H-(reaction occurred intensely), M(mol).

Discussion

There are two different substrates in peroxidase reaction, namely, hydrogen acceptor and hydrogen donor. In the leuco-dye-peroxide

reaction introduced by Li son (1936), the hydrogen acceptor is hydrogen peroxide and the hydrogen donor is leuco-dyes or so-called

zinc leucos. The dyes utilized for this method were acid fuchsin

(L i s o n, 1936), patent blue V (L i s o n, 1938), and cyanol (D u n n,

Some Problems on the Leuco-Dye-Peroxide Reaction 295

1946). Recently, Mitsui and S h i n o d a (1966) also stated a use-fulness of eosin for the leuco-dye-peroxide reaction. Then, it can

be said that the dyes applicable to the leuco-dye-peroxide reaction are acid dyes in general. In these methods, hemoglobin and other reaction-positive materials stain very intensely almost immediately, in the shade of the original dye.

It was revealed by the present investigation that leuco-phloxine

(erythrosin BB)-peroxide solution is also able to demonstrate erythro-cytes and other materials. It is of interest that acid fuchsin, patent blue V, cyanol and phloxine show a similar type of triphenyl dye. However, it should be noted that the leuco-dye-peroxide reaction is originally of a different nature from the ordinary histochemical

peroxidase reaction. In general, the peroxidase reaction occurs most intensely at the

adequate concentration of hydrogen peroxide in the reagent. In the ordinary histochemical peroxidase reaction, the optimal concentration. of hydrogen peroxide is 0.005-0.01mol in the case of myeloid leuco-

cytes (for instance, one drop of 3 per cent hydrogen peroxide is added to 10 ml reagent). With increase of the concentration of hydrogen peroxide in the reagent, the peroxidase reaction of the leucocytes generally becomes weaker, and finally the reaction is almost abolished at the concentration of over 0.1-0.2 mol of hydrogen

peroxide (T s u k am o t o 1952, Mitsui et al. 1955). In the original method of the leuco-dye-peroxide reaction introduced by L i s o n

(1936), 1 ml of 3 per cent hydrogen peroxide is added to 10 ml of original leuco-dye solution. This concentration of hydrogen peroxide is much higher than the optimal value for the peroxidase reaction mentioned above. Furthermore, in the original method, the reagent contains a large amount of acetic acid, and the pH value of the reagent is about 2.0. This pH value is far lower than the optimal

pH value for the peroxidase reaction. Under these conditions, namely, with the reagent containing a large amount of hydrogen

peroxide and of acetic acid, erythrocytes are clearly stained, while neutrophil leucocytes are not stained at all.

Dunn and Thompson (1946), using patent blue method , reported that hemoglobin and the granules in certain leucocytes , particularly eosinophils stain distinctly, and that the patent blue stain is essentially a peroxidase type of reaction . However, this conclusive statement is doubtful. It is of interest that F a u t r e z and Lambert (1937) reported that erythrocytes and eosinophil leucocytes were distinctly stained with cyanol solution alone at pH

296Tadao Mitsui, Toshio Shinoda and Hiroshi Ogawa

6.4. Lillie and Bur t n e r (1953) identified the inhibitory action

of methanol, reporting that both the cytochemical peroxidase and Winkler-Schultze oxidase reactions were entirely abolished by one hour exposure to pure methanol at 25°C. Further, Sasaki (1952) stated that the benzidine-peroxide staining of leucocytes ended in failure by fixation with methanol for only three minutes. It was found by the present study that the leuco-dye-peroxide reaction of eosinophil leucocytes was not inhibited at all by methanol, whereas the benzidine-peroxide reaction of the same leucocytes were intensely inhibited by methanol. This is one of the big differences between the leuco-dye method and the ordinary peroxidase reaction.

In addition to this, it was revealed that the leuco-dye-peroxide reaction of eosinophil leucocyte was not inhibited even by heating to 100°C for ten minutes. Mitsui et al. (1955) reported that orthophenylenediamine-peroxide and benzidine-peroxide reactions of leucocytes were entirely abolished by the treatment with boiling water for five minutes. Therefore, it is questionable whether the leuco-dye-peroxide reaction under the conditions of the histochemical experiment is due to enzymatic action or to a nonprotein catalyst.

Mitsui et al. (1953) compared inhibitory actions upon the cytochemical peroxidase reaction between potassium cyanide and potassium hydroxide, and considered that alkalinity of potassium cyanide played the main role in this inhibitory action. The inhibi-tion test in the present study has revealed that the leuco-phloxine-peroxide reaction of the eosinophil leucocytes has a stronger resistance to potassium hydroxide than the benzidine-peroxide reaction. This is probably due to the difference in the staining properties between the two reactions. It was already reported by Mitsui et al. (1953) that the benzidine-peroxide reaction of eosinophil leucocyte is not inhibited by one molar solution of potassium cyanide. In the present study a similar result has been obtained in the experiment with the leuco-phloxine-peroxide reaction. However, it is worthy of special comment that the leuco-phloxine-peroxide reactions of eosino-phil leucocyte and neutrophil leucocyte are rather activated by one molar solution of potassium cyanide. Such a phenomenon can never be observed in the ordinary benzidine-peroxide reaction. The mecha-nism of this peculiar activation by potassium cyanide is not obvious.

Some Problems on the Leuco-Dye-Peroxide Reaction291

Conclusion

It was revealed by the present study that phloxine (erythrosin BB) can be used for the leuco-dye-peroxide reaction in place of patent blue V. The leuco-phloxine-peroxide solution stains erythrocytes, eosinophil leucocytes and horse-radish tissues deeply red. These cells and tissues are similarly stained by simple phloxine solution alone. However, the color produced in these cells and tissues by the leuco7phloxine-peroxide solution is more stable to various solvents than that produced by the simple phloxine solution.

The reagent of the leuco-dye method is generally quite acid and this pH value is far lower than the optimal pH value of the ordinary

peroxidase reaction. The leuco-dye-peroxide reaction does not occur in neutrophil leucocytes nor in monocytes. Furthermore, the leuco-dye- peroxide reaction is not inhibited by methanol, high alkaline solution nor by the treatment with 100°C for 10 minutes.

Judging from these data it can be concluded that the leuco-dye method is of a quite different nature from the ordinary histochemical

peroxidase reaction. In other words, the leuco-dye method probably demonstrates a pseudoperoxidase reaction.

References

D u n n, R. C. : Arch. Path. Vol. 41, p676-677, 1946. D on n, R. C. and Thompso n, E. C.: Stain Technology. Vol. 21, p65, 1946. F a u t r e z, J. et L a mber t, P. P.: Bull. Histol. Tech. micr. Vol. 14, p29, 1937. Lilli e, R. D. and Bur t n e r, H. J.: Journ. of Histochem. and Cytochem. Vol. 1, p8,

1953. L i s o n, L.: Histochimie animale. Gauthiers Villars, Paris, 1936. Liso n, L.: Beit. path. Anat. Vol. 101, p94, 1938. Mitsu i, T., Mar u y a m a, S. and K a m e z a w a, S.: Okajimas Fol. anat. jap. Vol.

25, p177, 1953. Mitsu i, T. and Ka m e z a w a, S.: Okajimas Fol. anat. jap. Vol. 27, p335, 1955. Mitsu i, T. and S h i n o d a, T.: Arch. histol. jap. Vol. 27, 071, 1966. S a s a k i, M.: Okajimas Fol. anat. jap. Vol. 24, p193, 1952. Tsukamot o, K.: Okajimas Fol. anat. jap. Vol. 24, p347, 1952.