Hydrochloric acid N

Transcription

1 ON THE FIXATION OF MUCIN AND THE PREPARATION OF AUTORADIOGRAPHS. By N. G. HEATLEY, D. W. JERROME, M. A. JENNINGGS and H. W. FLOREY. From the Sir William Dunn School of Pathology, Oxford. (Received for publication 10th December 1955) THE preservation of the droplets of mucin, or what is sometimes called mucinogen, in mucus-forming epithelial cells, so that a histological and cytological picture approximating to the appearances in life are preserved, has for a long time been a subject of research and much controversy has occurred over the best methods to be adopted. Krause [1927] published a review of methods that had been used. The present communication records certain technical procedures, one of which is new, which may help those who wish to correlate function with structure to produce better specimens than is usual with mucous cells. It is not claimed that the use of any of the following methods will invariably produce good results. Much depends on the freshness of the tissue to be fixed and on other factors which it has so far proved impossible to define. EXPERIMENTAL Fixation and Embedding Fixation.-As a working hypothesis it was suggested that since swelling of mucin is the chief bugbear in fixation, substances that precipitate mucin might be helpful ingredients of fixatives. The effect of a number of protein precipitants was examined in the test tube on dialysed mucin obtained from a duodenal fistula of a pig. Of the reagents tested, phosphomolybdic acid gave a markedly more compact precipitate than did the others, but only if the ph was below about 3. When the precipitate was washed copiously with water it swelled in the course of an hour or two and became somewhat tenuous, but in 50 per cent ethanol this did not occur. After preliminary trials of tissue fixation in watery and ethanolic phosphomolybdic acid a mixture with the following composition was adopted and has been used with good results for about three years: Phosphomolybdic acid.. 5 per cent (w/v) Hydrochloric acid N Formalin per cent (v/v) of "40 per cent" Ethanol per cent (v/v) 124

2 Fixation of Mucin and Preparation of Autoradiographs In practice a stock solution is prepared containing 25 per cent (w/v) phosphomolybdic acid in 6-25N HCl. This keeps indefinitely, but the fixative is freshly prepared each day it is required (though this may not be necessary) by mixing 20 ml. of the stock solution with 65 ml. ethanol, 10 ml. of "40 per cent" formalin and 5 ml. water. Some batches of phosphomolybdic acid have given worse fixation than others, but this could not be correlated with UV absorption spectra or any other properties that were investigated. The success as a fixative did not appear to depend on the extent to which the stock solution became green or blue on standing, nor did ageing of the stock solution appear to improve or impair its performance. Analar 1 material gave better results on the whole than the various samples of ordinary laboratory grade material that were tested. The formalin was added in the hope of improving penetration of the phosphomolybdic acid, and at one time 5 per cent of the commercial solution of Teepol was added for the same reason, but this was afterwards omitted as it could not clearly be shown to confer any benefit. The method of using this fixative is as follows: (1) Immerse the tissue in the fixative immediately after removing from the animal. (2) After about hours' fixation transfer to 70 per cent alcohol, and change two or three times over a period of hours. (3) Dehydrate, clear and embed. We have generally worked with thin tissues such as the intestines of small animals or dissected mucosae of larger ones, and have no experience of the penetration of thicker blocks of tissue by this fixative. During fixation the tissue develops a greenish colour, and though this may be confined to the outer layers it does not seem to denote the limit of effective penetration. As a matter of convenience fixation has generally been for an overnight period, but for this type of material about 8 hours' fixation is sufficient. Trimming, if necessary, should be carried out after the washing in 70 per cent alcohol; if done while the tissue is still in fixative the cut surface will be stained blue (due to molybdenum blue?) from contact with the knife, and this will be evident to a varying degree in the stained sections. Embedding.-A very considerable improvement in results was obtained by embedding in a modified ester wax mixture recommended to us by Mr. E. H. Leach, developed by him from the method of Steedman [1947]. A further modification of this method giving even better results will be published in the near future [Chesterman and Leach, 1956]. The preservation of the tissues generally, and of mucin droplets in particular, was much better after embedding in this mixture than I The British Drug Houses Ltd. 125

3 126 Heatley, Jerrome, Jennings and Florey when paraffin was used-a finding that confirmed Mr. Leach's experience with Zenker-formol fixed material. The modified ester wax has the following composition: Diethylene glycol monostearate, purified g. Diethylene glycol distearate g. Castor oil ml. The mixture is placed overnight in the 500 C. oven to melt, then filtered. It should be removed from the oven when not in use. The embedding procedure is as follows: (1) Dehydrate in increasing grades of alcohol. (2) Pass through 3 or 4 changes of absolute alcohol. (3) Transfer to a mixture of equal volumes of absolute alcohol and benzene. (4) Clear in benzene. (5) Drain off as much benzene as possible and transfer to the molten modified ester wax mixture in the 500 C. oven. Pass through at least two changes of the mixture, the total infiltration time being usually hours. (6) Cast tissues in Leuckhart's brass angle pieces and place in a refrigerator to cool. (7) Trim blocks with a warm knife. If possible the blocks should be left for at least 24 hours before sectioning. Sections are mounted on slides by the hot plate method, never allowing the wax to melt, and are left on the hot plate to dry, then placed in the C. incubator overnight. Modified ester wax, although of lower melting point, is considerably harder than paraffin wax. As an embedding medium it appears to be generally superior in its completeness of infiltration and the greater support given to the tissue. Cutting is just as good, if not better; thin sections are easily cut and show very little compression; ribboning is excellent. In warm weather sectioning becomes difficult though not impossible; cooling of blocks seems less efficacious than in the case of paraffin. Staining.-Before staining, the ester wax is removed with xylol in the ordinary way and the sections are taken down to water, placed in 2 per cent sodium bisulphite for min., and washed in tap water for 5-10 min. The wash in sodium bisulphite largely prevents the development of molybdenum blue (?) which otherwise, particularly if haematoxylin is used, may darken the sections and obscure the staining. It is not needed if haomatoxylin staining is preceded by the periodic acid-schiff procedure. Mucicarmine and the periodic acid-schiff staining method both give excellent results on tissues fixed in this way. 1 The Watford Chemical Co., 30 Baker Street, London, W. 1, supply purified material suitable for this purpose. 2 Material from The British Drug Houses Ltd. has been found satisfactory.

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5 Fixation of Mucin and Preparation of Autoradiographs 127 Comment on Results Most of our work has been upon the gastro-intestinal tract. With the combined use of the phosphomolybdic acid fixative and embedding in modified ester wax it is usually possible to get excellent preservation of the mucin within the theca of the goblet cells of the small and large intestines. The contents of the theca appears in the form of fine granules, without swelling of the mucin or distortion of the cell. This is particularly true of the small intestine where the mucin is for unknown reasons more easily prevented from swelling than it is in the colon. Results were good in all the species from which tissues were used-cat, rat, mouse, rabbit and guinea-pig. (We must, however, expect the second cell type in the rabbit colon which, though apparently containing mucin, is no better preserved by this than by other fixatives.) A comparison with Zenker-formol fixation and with paraffin embedding is made in figs Other pictures illustrating the comparison with Zenker-formol and formol-saline fixation appear in Florey [1954]. The specimens shown in figs were all stained by the periodic acid-schiff method and have been magnified x FIG. 1.-Goblet cell. Rat. Small intestine. Fixed phosphomolybdic acid mixture, embedded in ester wax. Shows mucin preserved as fine granules evenly dispersed throughout the theca. FIG. 2.-Goblet cell. Rat. Small intestine. From the same rat as fig. 1. Received the same treatment except that specimen was embedded in paraffin. The mucin granules are preserved but they are not so evenly dispersed as in material embedded in ester wax. FIG. 3.-Goblet cell. Rat. Small intestine. Fixed in Zenker-formol and embedded in ester wax. The granules above the nucleus can be seen but those in the theca are not discrete as in figs. 1 and 2. FIG. 4.-Goblet cell. Rat. Small intestine. Treatment as for fig. 3 but embedded in paraffin. Discrete granules can be discerned above the nucleus but in the theca a fine meshwork represents the outlines of swollen droplets. FIG. 5.-Goblet cell. Cat. Small intestine. Fixed in phosphomolybdic acid mixture and embedded in ester wax. The mucin has been fixed as granules though these are not so fine as those in the goblet cell of the rat. FIG. 6.-Goblet cell. Cat. Small intestine. Fixed in Zenker-formol and embedded in ester wax. It is not possible to distinguish individual granules in the goblet cell. FIG. 7.-Goblet cell. Rat. Colon. Fixed phosphomolybdic acid mixture and embedded in ester wax. Mucin is preserved as granules. FIG. 8.-Goblet cell. Rat. Colon. Fixed as fig. 7 but embedded in paraffin. The mucin has been preserved as granules which are not so clearly seen as those in fig. 7. FIG. 9.-Goblet cell. Rat. Colon. Fixed in Zenker-formol and embedded in ester wax. Only a few individual granules of mucin are distinguishable. FIG. 10.-Goblet cell. Rat. Colon. Fixed as fig. 9 but embedded in paraffin. The mucin has swollen so much as to become a network.

6 128 Heatley, Jerrome, Jennings and Florey The superficial epithelium of the stomach is well preserved by the phosphomolybdic acid mixture, but only occasionally are the mucoid neck cells or the pyloric or Brunner's glands well fixed; in these, Zenkerformol fixation usually gives better preservation of the mucin. Peptic cells are disrupted in the phosphomolybdic acid mixture, but otherwise it proved to be a good general fixative of the tissues used, preserving, for instance, the basement membrane extremely well. A smaller experience with trachea and gall-bladder from the same species has shown excellent fixation of mucin in the epithelium of the gall-bladder and in the columnar cells and mucous glands of the trachea. Autoradiographs By the use of the phosphomolybdic acid mixture and Zenker-formol it has been possible to prepare tissues from stomach to colon in which mucin droplets do not swell and in which consequently any radioactive material that may be incorporated in the secretion is not artificially dispersed. Autoradiographs were made from the tissues of a series of animals that had been injected with Na235SO4. A comparison was made with material fixed in formol saline and it did not appear that the two metallic fixatives caused artefacts in the autoradiographs when a thin layer of celloidin was interposed between the section and the photographic emulsion. The method was as follows: (1) Mount sections on slides with Mayer's glycerin albumen, dry overnight, dewax, and take down to water. (2) Immerse in 2 per cent sodium sulphate for 3 hours to wash out free - 3SO42- and leave only " incorporated " 35S in the tissues. (3) Wash in tap water for 15 min. (4) Stain with the periodic acid-schiff procedure [Gomori, 1952]. (5) Counterstain, if required, with the combination used by Lendrum [1949] for nuclear staining (celestin blue followed by Mayer's haemalum), or with metanil yellow, or with both. (6) Cover with 0.5 per cent celloidin in the usual manner. (7) Rinse in distilled water. (8) In the dark room attach stripping film emulsion 1 to the section [Doniach and Pelc, 1950]. (9) Place slides in the dark drying box (fig. 11) till completely dry (usually 1-1k hours). (10) Transfer slides to light-tight, metal-lined boxes and place in the refrigerator at 40 C. for the required period of exposure of the film. In our work useful exposure times ranged from 2 to 12 weeks. 1 "Kodak" Scientific Plates, autoradiographic.

7 Fixation of Mucin and Preparation of Autoradiographs 129 (11) At the end of exposure develop in "Kodak" D 19b developer. Fix, wash in tap water for min., rinse in distilled water and dry completely in the drying box. (12) Remove overlapping film from the back of the slide. (13) Immerse in 60 per cent alcohol, absolute alcohol and xylol successively, 10 min. in each. (14) Mount in damar. j FIG. 11.-Dark drying box. The motor-driven centrifugal fan a blows air at room temperature through the circuit of the box in the direction indicated, i.e. through the removable filter b, around light traps c, along slide-containing chambers d and e, around light traps f, to leave the box through hole g. The inside of the box is painted flat black, and the overlapping back and lid h (which is closed by a spring-loaded clip j) are both completely lined with j inch foam rubber sheet which presses against the edges of shelves, etc. This construction ensures effective light-proofing even if slight shrinkage or warping of the wood should occur. Filter b consists of four layers of surgical gauze on a fine wire mesh support, clamped between metal frames. The slide carriers k, of Perspex, are so arranged that the current of air has maximum access to the slides. Examples of autoradiographs obtained by the methods described will be found in the following paper [Jennings and Florey, 1956].

8 130 Heatley, Jerrome, Jennings and Florey CONCLUSION AND SUMMARY 1. Phosphomolybdic acid, a mucin precipitant, is a useful ingredient in a histological fixative designed for the preservation of intracellular mucin. By its use the mucin within goblet cells and some other mucous cells can be preserved in the form of fine, compact granules, with little distortion of the theca or other cell structures. 2. This effect is enhanced by embedding in a modified ester wax mixture, which apparently penetrates and supports the tissue better than paraffin wax. 3. Using sections prepared in this way, autoradiographs have been obtained in which the sites of radioactivity can be related with some degree of accuracy to the mucin within each cell. REFERENCES CHESTERMAN, W. and LEACH, E. H. (1956). [In preparation.] DONIACH, I. and PELC, S. R. (1950). Brit. J. Radiol. 23, 184. FLOREY, H. W. (1954). In Lectures on General Pathology. Ed. H. W. Florey. London: Lloyd-Luke (Medical Books) Ltd. P GOMORI, G. (1952). Microscopic Histochemistry. University of Chicago Press. JENNINGS, M. A. and FLOREY, H. W. (1956). Quart. J. exp. Physiol. 41, 131. KRAUSE, R. (1927). Enzyklopiidie der Mikroskopischen Technik. 3. Berlin and Wien: Urban & Schwarzenberg. LENDRuM, A. C. (1949). J. Path. Bact. 61, 443. STEEDMAN, H. F. (1947). Quart. J. micr. Sci. 88, 123.