A study of the histochemical demonstration of cytochrome oxidase

Transcription

1 497 A study of the histochemical demonstration of cytochrome oxidase By R. G. BUTCHER, J. V. DIENGDOH, and J. CHAYEN (From the Department of Pathology, Royal College of Surgeons of England, Lincoln's Inn Fields, London, W.C.2) With 2 plates (figs. 1 and 2) Summary Burstone's procedure for the histochemical demonstration of cytochrome oxidase has been studied and applied to sections prepared by freezing in hexane and cutting by the controlled-temperature freezing-sectioning technique. The method has been modified by the inclusion of a treatment with Lugol's iodine to yield a stronger colour, which is stable for at least several weeks and which localizes the enzyme activity more precisely. The variants of the reaction have been compared in their effect on cardiac muscle, liver, and kidney of the rat. Introduction THE original 'G-Nadi' reaction was unsatisfactory because it produced a weak stain which faded rapidly and which was not localized precisely at the site of the enzyme reaction. Burstone's (1959, i960) detailed investigations resulted in considerable improvement in the histochemical demonstration of this enzyme. To avoid inhibition of this enzyme it was necessary to perform the reaction on unfixed tissue (Burstone, 1959) and hence a modification of the Adamstone- Taylor method was used (Adamstone and Taylor, 1948). However, in view of the considerable superiority of sections prepared by a newer preparatory method (Aves and Chayen, in preparation), it was decided to examine the reaction on such sections. Moreover, the method described by Burstone required testing more critically both as regards the precise localization of the stain and as regards its fading. The reasons for the exact sequence of events within the procedure, and for the concentration of some of the components, were not apparent. Lastly, the fact that iodine could be used to render the 'M-Nadi' reaction more permanent (Lison, 1936) deserved further study in relation to the 'G-Nadi' reaction. Materials and methods Male albino rats fed on a complete diet (M.R.C. rat diet B41) were killed by placing them under a funnel through which nitrogen was passed. Small portions of kidney, liver, and cardiac muscle were frozen in hexane at 70 C and placed in tubes which had been chilled with carbon dioxide ice for at least 18 h (Aves and Chayen, in preparation). Sections were cut at 8 p by the controlled-temperature freezing-sectioning technique (Cunningham and others, 1962) and mounted on clean slides. [Quart. J. micr. Sci., Vol. 105, pt. 4, pp , 1964.] L 1

2 498 Butcher, Diengdoh, and Chayen The incubation medium was prepared as follows (Burstone, 1959): 10 mg of N-phenyl-p-phenylenediamine and 10 mg of i-hydroxy-2-naphthoic acid were placed in a flask and dissolved in 0-5 ml of absolute ethanol. 35 ml of distilled water were added, followed by 15 ml of 0-2 M tris buffer ph 7-4 (24-2 g tris-(hydroxymethyl)-aminoethane in 170 ml in HCl and sufficient water to make a final volume of 1 litre). The solution was shaken and filtered through Whatman No. 1 filter paper into a Coplin jar. As a control, o-ooi M sodium azide was added to an identical incubation medium. Unfixed frozen sections were incubated either in the test or in the control solution for 60 min at room temperature. Too much autoxidation of the reactants occurred at higher temperatures and these were therefore avoided. After incubation, slides were transferred to a 10% solution of cobalt acetate in 10% formalin for 60 min. They were then washed in distilled water and mounted in Farrants's medium. The other variants on the procedures, and the use of Lugol's iodine, will be described in the text. Results The effect of Burstone's procedure on frozen sections from hexane-chilled tissue In liver the reaction produced a weak stain which was stronger periportally than towards the centre of the lobules. The nuclei were unstained, the cytoplasm was coloured, and darker bodies could be discerned scattered throughout the cell (fig. 1, A). The kidney stained moderately, some tubules reacting more than others. Nuclei were unstained while separate granules, and possibly filaments, were seen in the cytoplasm of those cells which stained more intensely (fig. 1, c). The fibres of the heart muscle stained moderately, some regions showing rather greater intensity (fig. 2, A). Very small granules, which appeared to line the fibrils, were appreciably coloured; these were assumed to be the sarcosomes. There was also a significant degree of colour on the fibrils. After the stained and mounted sections had been kept for about one month in a covered receptacle, there was very little stain left in those of liver. Although the stain in the kidney sections had faded appreciably, granular particles were FIG. 1 (plate), A, section of rat liver stained by the Burstone procedure and photographed on the day of preparation. The cytoplasm is coloured, with darker areas of granular activity. B, rat liver stained as in A but treated with Lugol's iodine before chelation with cobalt acetate. Photographed on day of preparation. The darkly-stained granules stand out against a weakly-coloured background. The nuclei are unstained. c, section of rat kidney cortex prepared by Burstone's method and photographed on the day of preparation. Some tubules have reacted more than others. D, a similar area from the same section, photographed one month later. The stain has faded considerably from the tubules, although some granular particles are still very clear. E, rat kidney cortex stained as in B above and photographed on the day of preparation. The tubules have stained intensely, with little or no background colour. The glomerulus is unstained. F, a similar area from the same section, photographed one month later. Very little fading of the stain has occurred during that time.

3 100/j FIG. I R. G. BUTCHER, J. V. DIENGDOH, and J. CHAYEN

4 : J 25/i FIG. 2 R. G. BUTCHER, J. V. DIENGDOH, and J. CHAYEN

5 Histochemical demonstration of cytochrome oxidase 499 still very clear (fig. 1, D); in the cardiac muscle, the stain had faded largely from the granules, but was well marked on the fibrils (fig. 2, B). The control sections, which were treated with azide, showed virtually no stain either in this procedure or in the different variations of it described below. The effect of altering the order of the procedure for preparing the incubation medium The preparation of the incubation medium was altered in the following ways. After dissolving the two substrates in absolute ethanol, (a) water was added before the tris buffer and the resulting solution was placed into a Coplin jar without filtering; (b) tris buffer was added before water and the resulting solution was filtered into a Coplin jar; (c) tris buffer was added before water and the unfiltered solution was poured into a Coplin jar. Sections were then treated in the same way as described above. It was found that in some cases the substrates did not completely dissolve in the ethanol and filtering therefore gave a clearer incubation medium. Despite this, all these variants gave results similar to those obtained with the original Burstone method (above). Burstone's method of preparing the incubation medium was retained therefore in the subsequent experiments. The effect of altering the ph and the concentration of the tris buffer Incubation media were prepared as described above, with a series of 0-2 M tris buffers at ph values ranging from 6-o to 9-0. The ph value of each of the media was checked just before use; it was found that the constituents did not alter the ph of the buffer. However, when this experiment was repeated with tris buffers of 0-05 M concentration, the constituents of the incubation medium were found to alter the ph of the final medium by about o-6 of a ph unit. It seemed necessary therefore to use the higher concentration of buffer to obtain adequate buffering. FIG. 2 (plate), A, section of rat heart stained by the Burstone procedure and photographed on the day of preparation. The very small granules are presumed to be the sarcosomes. The fibrils are also well stained. B, a photograph taken one month later of a similar area from the same section. The granular staining has largely faded, while the fibrils are still appreciably coloured. C, rat heart treated with Lugol's iodine after incubation and before chelation. Photographed on day of preparation. The sarcosomes have stained intensely, while the fibres are almost uncoloured. D, another area from the same section photographed one month later. Note the lack of activity on the intercalated disc. E, a photograph of the same section at higher magnification, one month after preparation. The granular staining of the sarcosomes is still as intense as on the day of preparation. F, rat kidney cortex treated with iodine between incubation and chelation, and photographed at higher magnification one month after preparation. The nuclei are unstained and darker granules can be seen in the region of the brush border.

6 500 Butcher, Diengdoh, and Chayen The activity of cytochrome oxidase in the tissues studied was found to be high over a ph range of 6-5 to 8-o, but there was little activity at ph values of 6-o and 8-5. The effect of varying the conditions of chelation After incubation, sections were placed in one of the following solutions for 60 min: (a) io% cobalt acetate in 10% formalin plus 5 ml 0-2 M acetate buffer ph 5-2 (Burstone, i960); (b) 10% aqueous solution of cobalt acetate; (c) 1 % aqueous solution of cobalt acetate; (d) distilled water. All sections were then washed in distilled water and mounted in Farrants's medium. No differences could be found as a result of using varying concentrations of cobalt acetate. Neither formalin nor acetate buffer had an appreciable effect. However, chelation with cobalt resulted in the stain being localized more precisely on mitochondria or similar structures, such as the sarcosomes of the cardiac muscle, and produced less diffuse cytoplasmic reaction. It also retarded the fading of the dye, although the colour was appreciably weaker even after one week, becoming progressively diminished. The effect of Lugol's iodine By analogy with its use in the 'M-Nadi' reaction, the iodine solution attributed to Lugol (1 g iodine, 2 g potassium iodide, and 100 ml distilled water) was used in an attempt to render the reaction more permanent. The following methods were employed: (a) after incubation, sections were transferred to Lugol's iodine for 2 min; (b) after incubation, sections were transferred to a 1 % aqueous solution of cobalt acetate for 60 min and then placed in Lugol's iodine for 2 min; (c) after incubation, sections were transferred to Lugol's iodine for 2 min and then placed in a 1% aqueous solution of cobalt acetate for 60 min; (d) sections were placed in a solution which contained 70 ml of distilled water and 30 ml of tris buffer (i.e. the incubation medium minus the substrates) for 60 min, and then immersed for 2 min in Lugol's iodine and subsequently for 60 min in a 1 % solution of cobalt acetate. All sections were then washed in distilled water and mounted in Farrants's medium. Sections incubated in a medium without substrates (d, above) and then treated with Lugol's iodine, showed only a slight background coloration and no specific staining. All the other sections which had been treated with iodine showed a more intense reaction than did the equivalent untreated sections. The colour was brown.

7 Histochemical demonstration of cytochrome oxidase 501 When the iodine was applied without chelation, the stain was more intense than that produced by Burstone's method alone. The reaction appeared not only on cytoplasmic granules in liver and kidney, and on the sarcosomes in the muscle, but also as a strong diffuse cytoplasmic yellowish-brown colour. After a month this diffuse colour had faded, leaving the separate particles very apparent on account of their almost black staining. A great deal of diffuse cytoplasmic staining was also observed when the sections were treated with iodine after the chelating procedure. Thus although the sarcosomes stained deeply, the muscle fibres were also stained, albeit a deep yellow-brown. The latter colour also faded after about one month, to leave the almost black sarcosomes with little background colour. In the kidney, although the glomeruli stained diffusely yellowish-brown, this faded within the month. The tubules stained with variable intensity: particles were almost black and did not fade, in contrast with the cytoplasm, which faded gradually. Similarly, sections of liver were coloured by an unstable diffuse yellowish-brown stain, in which dark cytoplasmic granules could be discerned. In all the tissues studied, when the treatment with Lugol's iodine preceded the chelation with cobalt, the diffuse cytoplasmic colour was very considerably reduced to a weak yellow. This background colour was not permanent, but even 3 months after the reaction had been performed, the cytoplasmic granules and organelles stained remained dark grey or black. Thus in the liver the cytoplasmic granules appeared clearly grey or black against a weak yellow background (fig. 1, B). In the kidney the granules stained very strongly, with a degree of orientation into what seemed to be filaments in the region of the brush border (fig. 1, E). A similar appearance was observed one month later (figs. 1 F; 2, F). Similarly, in the heart muscle, whether on the day that the reaction was performed (fig. 2, c), or one month later (fig. 2, D, E), the sarcosomes stained intensely, with little or almost no colour in the fibres themselves. The effect of sodium thiosulphate These experiments were repeated with the following modification: after treatment with iodine, sections were placed in a 5 % aqueous solution of sodium thiosulphate for either 4 or 30 min. The time of exposure to this solution did not affect the final result, which was similar to that of leaving the stained sections for some time. Thus the background colour appeared to be diminished, but since the colour changed from brown to grey, it was difficult to be sure that it was not a change of hue rather than a true fading that was the significant factor. Similarly the colour of the particles changed from brown to grey or black. However, their intensity was not diminished. Discussion Burstone made great advances in the histochemical demonstration of cytochrome oxidase activity. However, on sections prepared by the newer method, the colour produced by his reaction faded markedly and did not correspond to

8 502 Butcher, Diengdoh, and Chayen Cytochrome oxidase the clear localization of the enzyme that was expected from biochemical evidence of its exclusively mitochondrial location. The results described in this communication indicated that the method of preparing the incubation medium suggested by Burstone was optimal, that the concentration of cobalt acetate did not appear to be critical, and that neither the formalin nor the acetate buffer for chelation appeared to be necessary. Lugol's iodine intensified the colour and stabilized it against fading. Surprisingly it also improved the localization. Biochemical fractionation shows that cytochrome oxidase is contained exclusively in the mitochondrial fraction, and the use of Lugol's iodine before chelation tended to restrict the dark colour, produced by this reaction, to granules which appeared to correspond to mitochondria. That the reaction which was observed was due to the action of cytochrome oxidase was shown by the fact that it was abolished in the presence of azide. The coloured product formed by treatment with iodine was apparently capable of being chelated with cobalt. This procedure appeared to be necessary in order to yield more exact localization of the dye and so to reduce the intensity of the background staining. It is assumed that the iodine acted as an oxidant rather than by iodination, since the stain was not removed or severely disturbed by treatment with sodium thiosulphate. Thus it would appear that the following procedure may yield a fairly strong and rather permanent colour that indicates the true localization of cytochrome oxidase. 1. Dissolve 10 mg of N-phenyl-/>-phenylenediamine and 10 mg of i- hydroxy-2-naphthoic acid in 0-5 ml of absolute ethanol. Add 35 ml of water and then 15 ml of tris buffer ph 7-4; shake, and filter the solution into a Coplin jar. For the control add o-ooi M sodium azide to an identical medium. 2. Incubate unfixed frozen sections in the test and control solutions for 60 min at room temperature. 3. Immerse in Lugol's iodine for 2 min and then treat with a 5% aqueous solution of sodium thiosulphate for 4 min. 4. Place in a 1 % aqueous solution of cobalt acetate for 60 min. Wash in distilled water and mount in Farrants's medium. The authors wish to express their thanks to Dr. L. Bitensky for much helpful discussion and to Mr. A. L. E. Barron for the photomicrography. References Adams tone, F. B., and Taylor, A. B., Stain Tech., 23, 109. Aves, E. K., and Chayen, J. (in preparation). Burstone, M. S., J. Histochem. Cytochem., 7, nz. i960. Ibid., 8, 63. Cunningham, G. j., Bitensky, L., Chayen, J., and Silcox, A. A., Ann. Histochim., Lison, L., Histochimie Animale. Paris (Gauthier-Villars).