STAIN TECHNOLOGY VOLUME 27 JULY 1952 NUMBER 4

MUCOSAL WHOLE MOUNTS OF RODENT FORESTOMACH' PERIHAN CAMBEL and MARTHA GOODWIN ADAMS, Cancer Research

Laboratory, University of Florida, Gainesville, Fla. Received for publication, Nov. 14, 1951

AssmcT.-The most convenient separation of the mucosa of the forestomach of rats and mice was secured when the opened stomach was pinned flat (mucosa up) to the bottom of a paraffined petri dish and immersed 1-3 hours in 0.5-1.0% oxalic or acetic acid at room temperature. The separated mucosa was spread on a spatula and immersed in fixing fluid. Staining, dehydration, clearing and mount- ing in a synthetic mounting medium were by standard methods as used for celloidin sections.

Although macerating fluids have been used for many decades to dissociate tissues, the first account of a successful separation of the epidermis from the dermis by maceration with dilute acetic acid found in the literature was that of Menschel (1925). Hoepke (1927) gave a drawing of epidermis removed from the dermis with 0.25% acetic acid, but with no other details of technic. Baumberger, Sunt- zeff and Cowdry (1942) successfully separated the epidermis from the dermis by various other agents. The chemicals used by these authors were Nl1 and N / 3 ammonium hydroxide, ammonium chloride, am- monium carbonate, boric acid buffers, disodium phosphate and N / 1 hydrochloric acid. Heat was found to enhance the loosening effect. Heat alone was also used for separation (2 minutes at 50°C). His- tological sections of the two separated sheets of skin layers showed that the separation obtained was complete and reliable. Later Cow- dry, Cooper and Smith (1947) worked out an acetic acid maceration method for separating epidermis from dermis. Their purpose was the study of aging of the skin in whole mounts.

By means of a separation technic, Cooper and Schiff (1939) dis- covered the mitotic rhythm in the epidermis of mice and humans. Because it was planned to study mitotic activity in the glandular stomach of rodents, an attempt to secure whole mounts of the gas- tric mucosa by means of some of the methods mentioned was made.

'Grateful achnowledgment is made to the Damon RunJon Memorial Fund for a grant that defrayed the cost of this investigation.

STAIN TECHNOLOGY, VOL. 27, No. 4, JULY 1952

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186 ST.4IN TECHNOLOGY

Although the desired separation of the glandular mucosa in the entire stomach was not achieved, the removal of the forestomach niucosa of rats and mice proved successful.

METHODS

Twenty-five albino rats (Sprague-Dawley-Holtzman) and 25 Swiss albino mice (Tumblebrook Farm) were used for this study. The animals were killed by neckstroke. The stomachs were removed and cut open along the larger curvature. Food or food traces present were removed in physiological salt solution so that the mucosal sur- face was perfectly clean.

Separation by Heat: The stomach was placed with its serosal sur- face on a warm plate heated to 50°C (Baumberger, Suntzeff and Cowdry, 1942). After 3 to 5 minutes a superficial mucosal incision was made with an iridectomy knife near the cut border of the fore- stomach. This border was held with fine forceps while the mucosa was removed gently by a scraping and pushing movement of the knife. The forestomach mucosa was thus separated as far as the ridge, a fold separating the forestomach from the glandular stomach (Cambel and Sgouris 1951; see Fig. l), and then cut off here and drbpped into saline solution to unfold. After unfolding, it was drawn up on a spatula and placed in the fixative (Formalin, 1/10 aqueous; Carnoy’s fluid; or 80% ethanol). After fixation the mucosal sheet was stained in aqueous alum hematoxylin or Weigert’s iron hematoxylin and eosin, dehydrated, cleared like a celloidin section, and mounted in a synthetic mounting medium.

Separation by Mucerutionr Paraffin dissecting dishes (Petri) were prepared. The stomach was pinned with its serosal surface on the paraffin. It was then immersed with macerating fluids. Various soh- tions of different concentrations were chosen for this purpose accord- ing to their polarity (Ray, 1947). Maceration was carried out at room temperature as well as in the cold room-at 4°C. The stomach was alIowed to remain in the macerating fluid untiI the forestomach mucosa floated free from the underlying submucosa. The time of separation was found to vary at room temperature from I to 3 hours with 0.5-I% oxalic acid, and with 0.5-1% acetic acid. Better and quicker separation was achieved at room temperature rather than in the cold room. In the cold room, a maceration of 24 hours as an average was required. Both 0.25% oxalic acid and 1.0% boric acid solutions proved too weak for a complete separation, even after macerating for 24 hours at room temperature. The time of macera- tion with acetic acid solutions at room temperature showed indi- vidual and age variations. Separation was more difficult in the older animals.

After separation, the epidermal sheets were fixed, stained, dehy- drated, cleared and mounted as previously described.

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WHOLE MOUNTS OF FORESTOMACH 187

DISCUSSION Separation was more easily accomplished in the mouse. The histo-

Iogical appearance of the stained whole mounts was very similar to those of the skin (Menschel, 1925; Hoepke, 1927; Baumberger et d., 1942; Cowdry et al., 1947; Cooper and Smith, 1939). In whole mounts of the rat’s forestomach mucosa fixed in Carnoy’s fluid, un- identified granules were observed in the squamous cells. These gran- ules stained blue with hematoxylin. Their sizes showed little varia- tion, so it was decided that they could not be identical with kerato- hyalin granules.

Paraffin sections of the separated sheets showed separation to be complete and reliable. It is felt that the whole mount method ap- plied to the forestomach of rodents, especially the mouse, will be useful in morphological and histochemical studies, including inves- tigations of mitotic activity and aging of this organ.

REFERENCES

BAuhfBERGm, J. P., SUNIZEFF, V., and COWDRY, E. V. 1942. Methods of the sepa- ration of epidermis from the dermis and some physiologic and chemical properties of isolated epidermis. J. Nat. Cancer Inst., 2, 413-22.

CAMBEL, P., and Scourus, J. 1951. Modifications in Bowie’s staining of pepsino- gen granules. Stain Techn., 26, 245-6.

COOPER, Z., and SCHIFF, A. 1939. Mitotic rhythm in human epidermis. Proc. Soc. Exp. Biol. and Med., 39, 323-4.

COWRY, E. V., CMWER, 2.. and SMITH, W. Program of research on aging of the skin. J. Gerontol., 2, 31-44.

HOEPKE, H. 1927. Die Haut. In v. Moellendorffs H a n d b . der Mikrmkop. Anat. des Menschen. Berlin, Julius Springer. Bd. III/I, p. 3.

MENSCHEL, H. 1925. Zur Kolloidchemie und Pharmakologie der Keratinsub- stanzen und der menschlichen Haut. Arch. Exp. Pathol. Pharmakol., 110. 145.

RAY, F. E. 1947. Organic Chemistry. J. B. Lippincott Co., Chicago, Rev. Ed., p. 161.

1947.

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