Vol. 48, No. 4
THE JOURNAL OP INVESTIGATIVE DERMATOLOGY
Printed in U.S.A.
1967 by The Williams & Wilkins Co.
EFFECT OF ELECTRON STAINS AND DESMOSOME; AN ELECTRON MICROSCOPIC STUDY* JINRO KOMURA, M.D. AND SHTGEO OFUJI, M.D.
The first description of the fine structure of
MATERIAL AND METHOD5
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.
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
ELECTRON STAINS AND DESMOSOME
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.
, tt't '
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).
ELECTRON STAINS AND DESMOSOME
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-
brane. In the glutaraldehyde-fixcd section, the
Karrer (10) and Fawcctt (11) state that
attachment plaques are intensely stained by
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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
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