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A new method for separation of the epidermis and dermis
BIOCHEMICAL
MEDICINE
12,
162-165 (1975)
A New of the
Method Epidermis
T. A. LAWSON Department
for
of Pathology,
University
Separation and
Dermis
AND A. W. POUND of Queensland.
Brisbane.
Australia
Received August 19, 1974
In experimental animals, most chemically induced tumors arise in the epidermis. Consequently studies on the biochemistry of skin carcinogenesis can be facilitated if epidermal preparations can be obtained free from dermis. Many methods for the separation of dermis and epidermis exist, involving the use of keratomes (l), stretching and scraping the skin (2), heating the skin (3), chemicals (4), and enzymes (5). In our hands these proved unsatisfactory. The yield of epidermis was small and the methods were too time consuming. One of the prerequisites of a suitable technique was that it had to be applicable to large numbers of samples almost simultaneously, i.e., about 20 in half an hour. Contact adhesives have also been used, in particular Evo-stik (Evode Ltd., Stafford, Great Britain) in the separation of human skin. Application of Scotch tape after tryptic digestion has been used to remove epidermis from hairless mouse skin (6). These were not reliable when used with furry mouse skin but led us to consider the adhesive used here, Eastman 9 10. METHODS Seven week old male Crackenbush mice were used. The hair on the back, from the forelegs to the tail, was clipped with fine electric clippers, or depilated with barium sulphide paste. Immediately after the animals were killed a rectangular piece of skin, about 2 x 3 cm was separated and cut along three sides of the rectangle so that it was still joined to the body along the fourth side. Stiff filter paper of similar proportions was placed under this flap of skin to facilitate handling. The flap was cut from the remaining skin and placed flat, epidermis down, onto a perspex microscope slide (about 8 x 2.5 cm) onto which had been smeared a uniform light coating of Eastman 910 adhesive (Eastman Chemical Corp., Kingsport. Tennessee, U.S.A.). Firm contact was established between the specimen and slide by pressing them between two clean slides for 20 to 30 sec. The filter paper backing was removed and the slide with adherent skin was incubated in aqueous acetic acid (1.5%; v/v). A note of caution should be raised con162 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SEPARATION
OF
THE
EPIDERMIS
AND
163
DERMIS
cerning the avidity with which this adhesive cements human skin to anything bearing it. After about 1.5 hr, the dermal tissue distal from the slide had become gelatinised and was readily but gently peeled off. Any vestigial gelatinised tissue was removed by gentle scraping with the back of a scalpel blade aided, if necessary, by a further ten minutes incubation in acetic acid. The tissue remaining on the slide was not gelatinised by the acetic acid and was scraped off with a scalpel blade. RESULTS AND DISCUSSION After incubation in aqueous acetic acid (1.5%; v/v) mouse skin was readily separable into two fractions, the gelatinised fraction (A) which was believed to be the dermis, and the tissue remaining on the slide (B) which was believed to be the epidermis. The separation was believed to occur along the line of connective tissue that lies between the dermis and epidermis. Determination of the hydroxyproline content (7) of the two fractions showed that 92 +- 7% of the dermal collagen was found in fraction A, substantiating the belief that the separation occurred along the dermal-epidermal junction. It further indicates that the extent of contamination of the tissue remaining on the slide by the gelatinised tissue (dermis) was very small. The tissue remaining on the slide was stained with haematoxyiin and eosin and was observed to be epidermis. Figure 1 shows a photomicrograph of a preparation of the epidermis stained with a routine haematoxylin-eosin method from a mouse that had been depilated with barium sulphide paste. The specimen shows the epidermis viewed from
FIG. dermis.
1. Preparation showing H.E. stain. x80.
view
of epidermal
basal
cell
layer
after
removal
of the
164
LAWSON
AND
POUND
FIG. 2. Photomicrograph of same preparation as in Fig. 1 showing area in which further scraping had removed the basal cell layer of the epidermis to expose the squamous cell layer. H.E. stain. x80.
the deep surface. The stained necks of the hair follicles project upwards from the epidermis, are out of focus and the removed hair leaves a hole. The hair roots have been removed with the dermis. There is no evidence of dermal collagen in this preparation. Figure 2 shows one-half of the same preparation in which the basal layer of the epidermis had been removed by light scraping with a scalpel blade, to expose the still adherent layer of flat squamous cells. There is a fair amount of debris from the cement. With heavier scraping the epidermis is removed completely, with some debris. Smears of the removed layers showed suspensions of cells, singly and in clumps also with some debris. In preparations from animals that had only been clipped, a considerable amount of hair is present between the slide and the epidermis which obscures optical detail. This hair binds the hair follicles complete with the hair roots to the preparation where they remain as fingerlike projections from the epidermis. It seems likely that further development might provide a method of considerable versatility. As the dermis was gelatinised it was readily homogenised and protein and DNA could be extracted (8). RNA could not be extracted. The epidermis was in the form of flakes of tissue and could only be homogenised in a more vigorous way, either by ultrasonication or in a motordriven bladed homogeniser (Silverson Machines) preferably after grinding in liquid nitrogen. Protein, DNA (8), and RNA (9) could be extracted. The method described here gives very pure preparations of dermis and epidermis that are suitable for determinations of the content of materials present in each. We have used them to determine the binding of ethyl
SEPARATION
OF
THE
EPIDERMIS
AND
165
DERMIS
carbamate-(2sH) to DNA and to examine the incorporation of thymidine”H into dermal or epidermal DNA (10). We doubt if these fractions could be used for in vitro metabolic studies, or studies of enzyme activities. SUMMARY A method is described for the separation of the dermis and epidermis in which the skin is stuck, epidermis down, to a microscope slide with the contact adhesive Eastman 910. Incubation in aqueous acetic acid (1.5%; v/v) allows the dermis to be peeled from the epidermis which can then be scraped from the slide. REFERENCES 1. Laerum. 0. D. .I. Invest. Derm. 52, 204 (1969). 2. Van Scott, E. J. J. Invest. Derm. 18, 377 (1952). 3. Baumberger, J. P., Suntzeff. V., and Cowdrey, E. V. J. Natl.
Cancer
(1942). 4. Felsher, Z. Proc. Sot. Exptl. Biol. Med. 62, 213 (1946). 5. Giovanella. B. C., and Heidelberger, C. Cancer Res. 25, 161 (1965). 6. Alevaikki, M. Acta Path. Microbial. &and. Suppl. 226 (1971). 7. Woessner, J. F. Arch. Biochem. Biophys. 93, 440 (1961). 8. Colburn, N. C., and Boutwell, R. K. Cancer Res. 26, 1701 (1966). 9. Alston, W. C., and Thompson, R. Y. Cancer Res. 28, 746 (1968). 10. Pound. A. W., and Lawson, T. A. J. Natl. Cancer Inst., in press (1974).
Inst.
2, 413.
MEDICINE
12,
162-165 (1975)
A New of the
Method Epidermis
T. A. LAWSON Department
for
of Pathology,
University
Separation and
Dermis
AND A. W. POUND of Queensland.
Brisbane.
Australia
Received August 19, 1974
In experimental animals, most chemically induced tumors arise in the epidermis. Consequently studies on the biochemistry of skin carcinogenesis can be facilitated if epidermal preparations can be obtained free from dermis. Many methods for the separation of dermis and epidermis exist, involving the use of keratomes (l), stretching and scraping the skin (2), heating the skin (3), chemicals (4), and enzymes (5). In our hands these proved unsatisfactory. The yield of epidermis was small and the methods were too time consuming. One of the prerequisites of a suitable technique was that it had to be applicable to large numbers of samples almost simultaneously, i.e., about 20 in half an hour. Contact adhesives have also been used, in particular Evo-stik (Evode Ltd., Stafford, Great Britain) in the separation of human skin. Application of Scotch tape after tryptic digestion has been used to remove epidermis from hairless mouse skin (6). These were not reliable when used with furry mouse skin but led us to consider the adhesive used here, Eastman 9 10. METHODS Seven week old male Crackenbush mice were used. The hair on the back, from the forelegs to the tail, was clipped with fine electric clippers, or depilated with barium sulphide paste. Immediately after the animals were killed a rectangular piece of skin, about 2 x 3 cm was separated and cut along three sides of the rectangle so that it was still joined to the body along the fourth side. Stiff filter paper of similar proportions was placed under this flap of skin to facilitate handling. The flap was cut from the remaining skin and placed flat, epidermis down, onto a perspex microscope slide (about 8 x 2.5 cm) onto which had been smeared a uniform light coating of Eastman 910 adhesive (Eastman Chemical Corp., Kingsport. Tennessee, U.S.A.). Firm contact was established between the specimen and slide by pressing them between two clean slides for 20 to 30 sec. The filter paper backing was removed and the slide with adherent skin was incubated in aqueous acetic acid (1.5%; v/v). A note of caution should be raised con162 Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SEPARATION
OF
THE
EPIDERMIS
AND
163
DERMIS
cerning the avidity with which this adhesive cements human skin to anything bearing it. After about 1.5 hr, the dermal tissue distal from the slide had become gelatinised and was readily but gently peeled off. Any vestigial gelatinised tissue was removed by gentle scraping with the back of a scalpel blade aided, if necessary, by a further ten minutes incubation in acetic acid. The tissue remaining on the slide was not gelatinised by the acetic acid and was scraped off with a scalpel blade. RESULTS AND DISCUSSION After incubation in aqueous acetic acid (1.5%; v/v) mouse skin was readily separable into two fractions, the gelatinised fraction (A) which was believed to be the dermis, and the tissue remaining on the slide (B) which was believed to be the epidermis. The separation was believed to occur along the line of connective tissue that lies between the dermis and epidermis. Determination of the hydroxyproline content (7) of the two fractions showed that 92 +- 7% of the dermal collagen was found in fraction A, substantiating the belief that the separation occurred along the dermal-epidermal junction. It further indicates that the extent of contamination of the tissue remaining on the slide by the gelatinised tissue (dermis) was very small. The tissue remaining on the slide was stained with haematoxyiin and eosin and was observed to be epidermis. Figure 1 shows a photomicrograph of a preparation of the epidermis stained with a routine haematoxylin-eosin method from a mouse that had been depilated with barium sulphide paste. The specimen shows the epidermis viewed from
FIG. dermis.
1. Preparation showing H.E. stain. x80.
view
of epidermal
basal
cell
layer
after
removal
of the
164
LAWSON
AND
POUND
FIG. 2. Photomicrograph of same preparation as in Fig. 1 showing area in which further scraping had removed the basal cell layer of the epidermis to expose the squamous cell layer. H.E. stain. x80.
the deep surface. The stained necks of the hair follicles project upwards from the epidermis, are out of focus and the removed hair leaves a hole. The hair roots have been removed with the dermis. There is no evidence of dermal collagen in this preparation. Figure 2 shows one-half of the same preparation in which the basal layer of the epidermis had been removed by light scraping with a scalpel blade, to expose the still adherent layer of flat squamous cells. There is a fair amount of debris from the cement. With heavier scraping the epidermis is removed completely, with some debris. Smears of the removed layers showed suspensions of cells, singly and in clumps also with some debris. In preparations from animals that had only been clipped, a considerable amount of hair is present between the slide and the epidermis which obscures optical detail. This hair binds the hair follicles complete with the hair roots to the preparation where they remain as fingerlike projections from the epidermis. It seems likely that further development might provide a method of considerable versatility. As the dermis was gelatinised it was readily homogenised and protein and DNA could be extracted (8). RNA could not be extracted. The epidermis was in the form of flakes of tissue and could only be homogenised in a more vigorous way, either by ultrasonication or in a motordriven bladed homogeniser (Silverson Machines) preferably after grinding in liquid nitrogen. Protein, DNA (8), and RNA (9) could be extracted. The method described here gives very pure preparations of dermis and epidermis that are suitable for determinations of the content of materials present in each. We have used them to determine the binding of ethyl
SEPARATION
OF
THE
EPIDERMIS
AND
165
DERMIS
carbamate-(2sH) to DNA and to examine the incorporation of thymidine”H into dermal or epidermal DNA (10). We doubt if these fractions could be used for in vitro metabolic studies, or studies of enzyme activities. SUMMARY A method is described for the separation of the dermis and epidermis in which the skin is stuck, epidermis down, to a microscope slide with the contact adhesive Eastman 910. Incubation in aqueous acetic acid (1.5%; v/v) allows the dermis to be peeled from the epidermis which can then be scraped from the slide. REFERENCES 1. Laerum. 0. D. .I. Invest. Derm. 52, 204 (1969). 2. Van Scott, E. J. J. Invest. Derm. 18, 377 (1952). 3. Baumberger, J. P., Suntzeff. V., and Cowdrey, E. V. J. Natl.
Cancer
(1942). 4. Felsher, Z. Proc. Sot. Exptl. Biol. Med. 62, 213 (1946). 5. Giovanella. B. C., and Heidelberger, C. Cancer Res. 25, 161 (1965). 6. Alevaikki, M. Acta Path. Microbial. &and. Suppl. 226 (1971). 7. Woessner, J. F. Arch. Biochem. Biophys. 93, 440 (1961). 8. Colburn, N. C., and Boutwell, R. K. Cancer Res. 26, 1701 (1966). 9. Alston, W. C., and Thompson, R. Y. Cancer Res. 28, 746 (1968). 10. Pound. A. W., and Lawson, T. A. J. Natl. Cancer Inst., in press (1974).
Inst.
2, 413.