Effect of Electron Stains and Desmosome; An Electron Microscopic Study*

Effect of Electron Stains and Desmosome; An Electron Microscopic Study*

Vol. 48, No. 4 THE JOURNAL OP INVESTIGATIVE DERMATOLOGY Copyright Printed in U.S.A. 1967 by The Williams & Wilkins Co. EFFECT OF ELECTRON STAINS ...

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Vol. 48, No. 4



Printed in U.S.A.

1967 by The Williams & Wilkins Co.


The first description of the fine structure of


the desmosome in the epidermis was recorded by Porter in 1954 (1). He made ob-

Normal human skin used in this study was removed under general anesthesia from the back of a 30-year-old man. The tissue was cut in small

servations on the epidermis of larvae of

Amblystoma punctotum. Later Selby (2) dem- pieces and quickly immersed in 1% osmium

onstrated the existence of the structure in human epidermis comparable to those described by Porter, and in 1958, Odland (3) presented a schematic drawing of the fine

tetroxide huffed to pH 72 with phosphate-sucrose-

buffer or in 5% glutaraldehyde buffered to pH 7.2 with phosphate. After fixation for two hours, the tissues were dehydrated in ethanol and embedded in Epon 812 as described by Luft (4). Sections were cut on a JUM-5 ultramicrotome

structure of desmosomes in human epidermis. There is now general agreement on the exist-

with glass knives. They were mounted on car-

ence of the layers described by Odland, but bon-coated grids and stained at room temperathere is some disagreement concerning the ture with lead hydroxide according to Watson origin of the attachment plaques and some (5) for 5 minutes or uranyl acetate saturated in confusion about the nomenclature of each 50% ethanol for two hours. The pictures were taken with a HU-IIA Hitachi electron microscope.

desmosome structure.

On the other hand, in recent years the use


of electron stains such as uranyl and lead salts has been a routine procedure in most

1) Fine structure of desmosome (Figs. 4, 5, 6): As described by Odland, desmosome in the

electron micrographs. However, there is little

cytochemical information about these electron stains. Some investigators have pointed out that the effects of these elecron stains are limited to a certain cellular component in-

Malpighian. layer consists of two electrondense plaques, and of three thin dense layers

information of electron stains.

fixation (Fig. 1)—desmosomes, tonofibrils, mitochondria, ribosomes and nucleus—are well pre-

and four layers of lesser density between two plaques. This finding is more clearly observadicating some cytochcmical specificity. It has ble in glutaraldehyde fixation than in osmium also been suggested that the chemical identifi- fixation. Odland (3) refers to the two plaques cation of cellular components could be ac- as "attachment plaques" and to the three thin complished by the use of suitable electron layers as "intercellular contact layer" (in the stains which attach with some discrimination center) and "intermediate dense layer." to the various chemical groups and lead to a 2) Glutaraldehyde fixation and uranyl acetate local increase in electron density. staining: After fixation in glutaraldehyde and As most electron strains are used as general staining by uranyl acetate, the membranous stains after osmium tetroxide fixation, the structures of the cells (the nuclear membrane, fixatives, e.g. glutaraldehyde, which do not of the outer membrane of mitochondria and the themselves introduce contrast, would be cytoplasmic membrane) are lacking (Fig. 2). more useful in obtaining the cytochemical Otherwise, the structures observed in osmium In this study, normal human epidermis fixed

in glutaraldehyde alone is stained by either served, and uranyl acetate serves as a general uranyl acetate or lead hydroxide to see the stain in glutaraldehyde fixation as it does in effects of electron stains. Some difference in osmium fixation. In the desmosome the increase the effects of these stains on desmosome struc-

in density of attachment plaques is remarkable after uranyl staining (Figs. 2 and 5). 3) Lead hydroxide staining: In the desmo-

ture is observed and the significance is discussed. ' From the Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan. Received for publication May 7, 1966.

some, the three thin layers between attach-


ment plaques are intensely stained, whereas the







Fic. 1. Standard electron micrograph of part of the Malpighian layer fixed fl 0504, embedded in Epon and stained on the section with uranyl acetate. X 21,000 N: nucleus,

M: mitochondria, T: tonofibril, m: melanin, cm: cell membrane, d: desmosome.

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FIG. 2. Partial view of the stratum Malpighii fixed in glutaraldehyde alone and stained

on the section with uranyl acetate. Note that no cell membrane is demonstrable. Desmosome

(d) are well preserved, and nucleus (N), ribosomes (R) and tonofibrils (T) are stained similarly. The outer membrane of mitochondria (M) is not visible. X 27,000 m: melanin.






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Fm. 3. Partial view of the stratum Malpighii fixed in glutaraldehyde alone and stained on the section with lead hydroxide. No cytoplasmic details are clarified with this stain except for ribosomes (R), which are remarkably stained, part of the desmosome (d) and the nucleus (N) which shows peripheral distribution of chromatin masses characteristic of the glutaraldehyde fixation. X 50,000

density of the attachment plaques is very the membranes are extracted during embedding low (Figs. 3 and 6). In the cytoplasm, ribo- by Epon and the presence of membranous elesomes are well stained and nuclear structure is

also stained. However, tonofibrils and mitoehondria are not clearly discernible (Fig. 3).

ments is not revealed.

In this study, the desmosome apparently

withstands Epon embedding and its fine structure is well preserved by glutaralde-

4) Density between the attachment plaques: The density of the four layers of lesser density hyde, although the cell membrane is not visibetween attachment plaques in osmium fixa- ble as described by Sabatini. tion is fairly uniform (Fig. 4), but in gluWhen Watson (5) introduced various heavy

taraldehyde fixation, the density of the layer metals for the staining of tissue sections, he between the attachment plaque and the inter- pointed out the possibility of staining reacmediate dense layer is almost translucent tions with some degree of specificity for submolecular differentiation. Since then the specificity of electron stains, especially uranyl and DISCUSSION lead salts has been discussed by many authors. Glutaraldehyde is a fixative introduced by Zobel and Beer (7), and Huxley and Zubay (8) Sabatini et al. (6) for enzyme cytocbemistry have demonstrated that DNA-containing proin electron microscopy. They noticed that the teins are preferentially stained by uranyl ace(Figs. 5 and 6).

glutaraldehyde fixed section presents a very different picture from the osmium tetroxide fixed section in that no membrane structure is discernible and that the chromatin material in the nucleus forms compact masses at the periphery. According to Sabatini, in the case of aldehyde fixation most lipid component of

tate. Daems and Persijn (9) have postulated that lead staining results in the staining of glycogen, increase in density of membranes and some

increase in density of ribosomes. The staining behavior of ribosomes with lead has also been stressed by various authors, e.g. Watson (5), Sabatini (6).



Fies. 4, 5 and 6. Higher magnification of the desmosome in the Malpighian layer. Fig. 4—0s04 fixation and uranyl staining, X 76,000: Fig. 5—glutaraldehyde fixation and uranyl staining, X 70,000. Fig. 6—glutaraldehyde fixation and lead staining, X 72,000. Desmosome consists of two dense plaques (p), two outer leaflets of the cell membrane or Odland's in-

termediate dense layer (il), a layer (X) referred to as the intercellular contact layer by Odland and of less electron dense substance between the layers. Intercellular contact layer

(X) is not remarkable in the osmium fixed section. (Fig. 4) Notice that the attachment plaques are intensely stained by uranyl acetate (fig. ),while the lead stain gives an increase in density of Odland's intermediate dense layer (il) and intercellular contact layer (X). (Fig. 6) The density of "the four layers of lesser density" between two plaques is uniform in 0504 fixation (Fig. 4), but not uniform in glutaraldehyde fixation (Figs. 5 and 6) as the

layer between the attachment plaque and the intermediate dense layer is almost translucent.

In the present study, lead hydroxide shows an intense affinity to ribosomes, nuclear struc-

tures arid part of the desmosomc structure. However, uranyl acetate seems to serve rather as a general stain in the glutaraldehyde fixed

section as it does in the osmium fixed sec-

Odland's attachment plaques seem more likely to be composed of a substance of cytoplasmic origin which is closely adherent to the irmer leaflet of the tn-laminar structure of unit mem-

brane. Karrer also states that Odland's new term, intermediate dense layer, would be un-

tion. In the desmosomc, some interesting dif- necessary because it is simply the outer leaflet ference between the effects of lead and uranyl of the cell membrane. Later these findings were stains is observed. For understanding, the fine confirmed by Farquhar and Palade (12). In structure of the desmosome of human epider- the human epidermis, Brody (13) described the outer surface of the attachment plaque as mis is briefly reviewed. Odland first recorded within the desmosome appearing to be formed by the cell membrane. In the present investigation, two electron several specialized electron-dense layers which he referred to as "attachment plaques, inter- plaques and three thin layers were noticed in mediate dense layers and an intercellular contact the desmosomc as originally described by Odlayer." According to Odland, the attachment land, most clearly in the glutaraldehyde-fixed plaque appears to be a specialized thicking of and uranium-stained section. In the osmiumthe cell membrane to which tonofilaments are fixed section, Odland's intercellular contact anchored. Later, the same layers have been con- layer is not remarkable, but it seems to have firmed in the cell interconnections of various no connection with the cell membrane. The inepithelia, but the relationship of cell membrane termediate dense layer seems to be continuof various layers within the dcsmosome has been

ous with the outer leaflet of the cell mem-

interpreted differently.

brane. In the glutaraldehyde-fixcd section, the

Karrer (10) and Fawcctt (11) state that

attachment plaques are intensely stained by



uranyl acetate, while the contrast of the three SUMMARY dense layers between the plaques is greatly 1) Electron microscopic findings of the enhanced by lead hydroxide. This shows that Malpighian layer and desmosome in normal Odland's intercellular contact layer is cyto- human epidermis after uranyl acetate and lead chemically close to the intermediate dense hydroxide staining on the osmium tetroxidc or

layer which is considered to be the outer glutaraldehyde fixed sections are presented.

leaflet of the cell membrane as it stains in a 2) The staining behavior in the dcsmosomc similar way to the intermediate dense layer, indicates that Odland's intercellular contact and that the attachment plaques are very dif- layer is cytochemically close to the outer ferent cytochemically from cell membranes, indicating their cytoplasmic origin. Judging from the nranyl-stained section, the

leaflet of the cell membrane and the attachment

plaques arc of cytoplasmic origin, while the substance responsible for the adhesion of two inner leaflet of the cell membrane within the cells exists as a "coating" of the outer leaflet dcsmosome seems to be located so closely to of the cell membrane and the intercellular conthe outer surface of the attachment plaque that tact layer. the inner leaflet and attachment plaque are fused to make one layer. The reason the inner REFERENCES leaflet is not visible after lead staining in the 1. Porter, K.: Observations on the submicroscopic glutaraldchydc-fixcd section is difficult to exstructure of animal epidermis. Anat. Rec., 118: 433, 1954. plain. Possibly as the inner leaflet is fused to C. C.: An electron microscope study of the attachment plaque in the desmosome, the 2. Selby, the epidermis of mammalian skin in thin mode of staining may correspond to that of sections. J. Biophys. Biochem. Cytol., 1: the attachment plaque. The intense staining of attachment plaques by uranyl acetate must be

interpreted carefully and we will need further information on the cytochemical interactions between uranium and cellular components. The adhesion of cells must be attributed to some chemical substance between the layers in the desmosome rather than to structural interconnections since no fibrils can be seen crossing

the intercellular space. The density of "the four layers of lesser density" between two attachment plaques in the osmium-fixed section is fairly uniform, but in the glutaraldchydefixed section, the density between the attachment plaque and the intermediate dense layer is almost translucent. This indicates that the substance between these two layers is osmio-

philic and possibly of a lipid nature, and that the substance responsible for adhesion of two cells exists possibly as "coating" at the

420, 1955.

3. Odland, G. F.: The fine structure of the interrelationship of cells in the human epidermis. J. Biophys. Biochcm. Cytol., 4: 529, 1958.

4. Luft, J. R.: Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol.,9: 409, 1961.

5. Watson, M. L.: Staining of tissue sections for electron microscopy with heavy metals: II. Application of solutions containing lead and barium. J. Biophys. Biochem. Cytol., 4: 727, 1958. 6. Sabatini, D. D., Bensch, K. and Barrnett, R. J.: Cytochemistry and electron microscopy: The

preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19, 1963. 7. Zobel, C. R. and Beer, M.: Electron stains: I: Chemical studies on the interaction of DNA with uranyl salts. J. Biophys. Biochem. Cytol., 10: 335, 1961.

8. Huxley, H. E. and Zubay, C.: Preferential staining of nucleic acid-containing structures for electron microscopy. J. Biophys. Biochem. Cytol., 11:

273, 1961.

9. Daems, W. Tb. and Persijn, J. P.: Section

staining with heavy metals of osmium-fixed and formal-fixed mouse liver tissue. J. Roy. Micr. Soc., 81:


Cell Res., Suppl. 8:

174, 1961.

199, outer surface of the outer leaflet of the cell 10. Karrer, H. E.: Cell interconnections in normal human cervical epitbelium. J. Biophs. Biomembrane and around the intercellular conchem. Cytol., 7: 181, 1960. tact layer. This "coating" substance seems to 11. Fawcett, D. W.: Intercellular bridges. Exp.

withstand Epon embedding in glutaraldehydc

fixation and the membrane structure in the dcsmosomc is preserved. It may be possible to

12. Farquhar, M. C. and Palade, G. E.: Cell junc-

tions in amphibian skin. J. Cell Biol., 26: 263, 1965.

identify exactly the cytochemical nature of 13. Brody, I.: The ultrastructure of the tonofibrils this "coating" substance in the dcsmosome by suitable electron stains.

in the keratinization process of normal human epidermis. J. IJltrastruct. Res., 4: 264, 1960.



linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.