British Journal of Dermatology (1975) 93, 623.
Ultrastructural study of elaunin fibres in the secretory coil of human eccrine sweat glands G.COTTA-PEREIRA,* F.GUERRA RODRIGO AND S.BITTENCOURT-SAMPAIO Laboratory of Cell Biology, Gulbenkian Institute of Science, Ociras, Portugal; Department of Histology and Embryology, Faculty of Medicine, State University of Guanabara and Department of Histology and Embryology, Federal University of Rio dc Janeiro, Brazil: Department of Dermatology, Faculty of Medicine, Lisbon, Portugal Accepted for publication 7 March 1975
SUMMARY
The basement membrane of the secretory coil of human eccrine sweat glands was studied by light and electron microscopy. With classical staining methods for the elastic system, elaunin ring fibres were observed in the basement membrane ofthe secretory coil. The ultrastructural study showed that elaunin fibres are formed by bundles of fibrotubulcs 10-12 nm in diameter surrounding aggregates of amorphous material. These fibres differ from elastic fibres which are thicker and have an amorphous material more homogeneous and abundant.
In previous work, the distribution and morphology of the elastic system in the hixman dermis were studied and the existence of three types of fibres (oxytalan, elaunin and clastic fibres) was demonstrated (Bittencourt-Sampaio & Cotta-Pereira, 1971; Rodrigo, Cotta-Pereira & Riches, 1973; CottaPereira, Rodrigo & Bittencourt-Sampaio, 1975). The most superficial are the oxytalan fibres; these depart from the elaunin plexus, which is located parallel to the dermo-epidermal junction and is connected with the elastic fibres ofthe reticular dermis. The ultrastructure of elastic fibres is well known (Greenlee, Ross & Hartman, 1966; Ross & Bornstein, 1969). They consist of an amorphous substance identified as elastin surrounded by the 'elastic fibre microfibrils'. Recently, we observed that the oxytalan fibres are formed by bundles of fibrotubules similar to those visualized in elastic fibres, but devoid of elastin (Cotta-Pereira et al., 1975). The fine structure of elaimin fibres is not yet known. Observing sections of human dermis by light microscopy, we found elaunin fibres in the basement membranef ofthe secretory coil of human eccrine sweat glands (Cotta-Pereira & Bittencourt-Sampaio, * Reprint requests should be addressed to Dr Gerson Cotta-Pereira, Departamento de Histologia e Embriologia, Faculdade de Ciencias Mcdicas, Universidade do Estado de Guanabara, Avenida 28 de Setembro, 87, fundos. Vila Isabel. Estado de Guanabara, Ciid. Postal 20 000, Brazil. t In this paper the term 'basement membrane' is used to name the extraccliular support ofthe basal surface of epirhelia as observed by light microscopy. 'Basal lamina' is applied to the dense filamentous sheet visualized as a constituent of the basement membrane by electron microscopy. 623
624
G.Cotta-Pereira et al.
1973). The purpose of this investigation was to study this area with the electron microscope in order to describe the fine structure of the elaunin fibres. MATERIAL AND METHODS
Skin fragments biopsicd from different areas of human volunteers were fixed in 10",, formaldehyde and embedded in paraflin. Histological sections were stained by the following techniques: Vcrhoeff's iron haematoxylin, Weigert's resorcin fuchsin, Unna-Tanzer's orccin, and Gomori's aldehyde fuchsin. All these techniques were preceded or not by an oxidation with 40",, peracetic acid (Pearse, 1968). The PAS technique after treatment with diastase was also used associated with the Weigert's resorcin fuchsin.
FIGURE I. Elaunin fibres wrapping the secretory coil of a sweat gland. Weigcrt's resorcin fuchsin method (x 720).
Other fragments were fixed for 3 h in a mixture of tannic acid-glutaraldehyde (TAG), containing 0-25",, tannic acid and 3'\, glutaraldehyde in Millonig buffer (pH ^ 7-4) (Futaesaku, Mizuhira & Nakamura, 1972). After postfixation in 2",, osmium tetroxide they were embedded in Epon. The ultrathin sections were studied in a Siemens Elmiskop IA at 80 kV, after staining with uranyl acetate and lead citrate. RESULTS
By light microscopy, using resorcin fuchsin, orcein or aldehyde fuchsin, an envelope of fibres was observed surrounding the sweat gland secretory coil (Fig. i). When the resorcin fuchsin method was preceded by oxidation with peracctic acid, the fibrous structures were enhanced and thinner fibres intermingled with the thicker ones were visualized. After Verhoeff's stain, with or without previous oxidation, no fibres were observed (Fig. 2). The association PAS-resorcin fuchsin showed a double stained membrane surrounding the secretory coils. While the diastase-resistant PAS-positive inner layer formed a continuous envelope around the
FIGURE 2. Secretory coil of a swear gland. Elastic fibres are present only tar from the gland farrow). They arc not seen in the secretory coil basement membrane. Verhoeff's iron haematoxylin ( x 284}.
FIGURE 3. Basement membrane of a sweat gland secretory coil. Longitudinal section of an elaunin fibre, formed by patches of dense amorphous material (arrow) among fibrillar bundles (star). BL = basal lamina. AS = amorphous sheath ( x 32,400).
FIGURK 4. Basement membrane of a sweat gland secretory coil. Cross section of an elaunin fibre. Note the amorphous substance scattered among fibrotubular bundles ( x 32,400).
FIGURE 5. Basement membrane of a sweat gland secretory coil. Note the cross sectioned fibrotubules 10-12 nm in diameter (arrows) ( x 64,800).
Elaunin fibres in human sweat glands
627
FIGURE 6. Longitudinal section of an elastic fibre. The amorphous component is compact and homogeneous. Some microfibrils are observed in the periphery of the fibre ( x 16,200).
gland, the resorcin fuchsin stained outer layer showed different aspects. In cross sections, it often appeared as a discontinuous ring. In oblique or in longitudinal sections it was represented by a rank of points or parallel rods. The electron microscopic study of the secretory coil basement membrane showed that it is formed of a basal lamina connected on its inner face with the serous and myoepithelial cells by half-desmosomes, and on its outer face with an amorphous and low electron dense sheath. This amorphous sheath has an irregular thickness and it is surrounded by collagen fibrils and by elastic-like fibres formed by patches of an electron dense amorphous substance in the core of fibrotubular bundles (Figs 3-5). This amorphous material is not so compact and homogeneous as it is in the elastic fibres (Fig. 6). Bundles of fibrotubules 10-12 nm in diameter and without amorphous material were also visualized.
628
G.Cotta-Pereira et al, DISCUSSION
Previously, it was demonstrated by light microscopy that the classical staining methods for elastic tissue could reveal in the human dermis oxytaian, elaunin and elastic fibres (Bittencourt-Sampaio & Cotta-Pereira, 1971; Cotta-Pereira et al, 1975). Gawlik (1965) considered these three types of fibres as the normal constituents of the elastic system of connective tissues. In the present work parallel ring fibres wrapping the secretory coil of sweat glands were demonstrated by resorcin fuchsin, orcein or aldehyde fuchsin but not by the Verhoeff's method. Fibres with similar tinctorial characteristics were revealed in tendons, fibrous and hyaline cartilage and elastic laminae of developing aortae (Gawlik, 1965). They were named 'elaunin fibres' by Gawlik (1965). On the basis of these tinctorial affinities, the sweat gland ring fibres are elaunin fibres, differing from elastic fibres because they do not stain with Verhoeff's method. Using resorcin fuchsin after oxidation with peracetic acid, thinner fibres binding the thick ring fibres were revealed. They have therefore the same tinctorial characteristics as the oxytaian fibres (Fullmer & Lillie, 1958; Gawlik, 1965). It is concluded that a mesh-work of elaunin and oxytaian fibres involves the secretory coil of eccrine sweat glands. The main difficulty in studying elastic fibres with conventional electron microscopic techniques has been the irregular staining of their components. The introduction of tannic acid added to glutaraldehyde was a progress (Futaesaku ct al, 1972) simultaneously improving the fine details of the connective fibrils and giving a satisfactory staining of the elastin. With this method a good reproducibility of results is obtained. We tried several concentrations of tannic acid and the best results were observed with 0-25",, added to 3",, glutaraldehyde in Millonig's buffer. With this technique, the electron microscopic study of the ring fibres in cross and in longitudinal sections showed that they are formed by bundles of parallel fibrotubules 10-12 nm in diameter surrounding aggregates of amorphous material. They differ from elastic fibres which are thicker and where the amorphous material is more homogeneous and abundant. In his studies on elastogenesis, Gawlik (1965) found that in elastic laminae of developing aortae the oxytaian fibres appeared first and were later substituted by elaunin and finally by elastic fibres. Based on those observations oxytaian fibres may be considered as the most primitive elastic fibres. In agreement with this, are previous observations with the electron microscope demonstrating that oxytaian fibres are reduced to the fibrotubular component of the elastic fibres (Cotta-Pereira et al, 1975). In fact, during elastogenesis the fibrotubular component is the first to appear and thereafter the accumulation of amorphous material is progressive until the mature elastic fibre is formed (Fahrenbach, Sandberg & Cleary, 1966; Greenlee et al, 1966). In light microscopy, the concept of the elaunin fibre is only based on tinctorial data. As the ring fibres of sweat glands have elaunin characteristics we studied them with the electron microscope in order to investigate their fine structure. We found that they have ultrastructurai characteristics between the oxytaian and the elastic fibres. The fibrotubular bundles are abundant and the amorphous component is sparse. Functionally, elaunin and oxytaian fibres have been related to mechanical resistance because they are found in places where mechanical stress is exercised (Fullmer, 1958; Gawlik, 1965). Perhaps their mechanical resistance is based on the fibrotubular component. This hypothesis is in accordance with the absence of elastin in oxytaian fibres and its scarcity in elaunin fibres. The function of the elaunin ring fibres in the sweat coil could represent a mechanical support for the gland, completed by the thinner oxytaian fibres linking the thick elaunin ring fibres. Another fact to be discussed is the general structure of the basement membrane in the secretory coil of human eccrine sweat glands. From our observations, the inner layer, PAS-positive after
Elauninfibresin human sweat glands
629
diastase treatment, corresponds in electron microscopy to the area formed by the basal lamina and the amorphous sheath surrounding it. The outer layer is therefore constituted by elaunin, oxytalan and collagen fibres. ACKNOWLEDGMENTS Dr Cotta-Pereira was partly supported by the State Secretary of Science and Technology, Guanabara, Brazil. The authors wish to thank Dr J.F.David-Ferreira for his valuable discussion and criticism. Technical assistance was provided by Mr J.C.Lemos, Mrs Maria Teresa Plantier and Mrs Maria da Rcssurrei^ao Alpior^a. Miss Maria Manuela Magalhaes typed the manuscript.
REFERENCES BtTTENCOURT-SAMPAio, S. & COTTA-PEREIRA, G . (1971) Distribui^ao das libras elasticas, elauninicas e oxicalanicas na derme superior em pele humana. Anais Brasileiros de Dermaiologia, 46, 333. COTTA-PEREIRA, G . & BITTENCOURT-SAMPAIO, S. (1973) Contribui^ao ao estudo da cstrutura da membrana basal de glandulas sudoriparas ecrinas. Anais Brasileiros de Dermatalogia, 48, 319. COTTA-PEREIRA, G., RODRIGO, F . G . & BITTENCOURT-SAMPAIO, S. (1975) Oxytalan, elaunin and elasUc fibers in the human skin. Journal of Invesiigative Dermatology. Submitted for publication. FAHRENBACK, W.H., SANDBERG, L.B. & CLEARY, E . G . (1966) Ultrastruccural studies on early elastogenesis. Anatomical Record, 155, 563. FULLMER, H . M . (1958) Differential staining of connective tissue fibers in areas of stress. Science., 127, 1240. FULLMER, H.M. & LILLIE, R . D . (1958) The oxytalan fiber: a previously undescribed connective tissue fiber. Journal of Histochemistry arid Cyiochertiistry, 6, 425. FuTAESAKU, Y., MizuHiRA, V. & NAKAMURA, H . (1972) A new fixation method using tannic acid for electron microscopy and some observations of biological specimens. Proceedings of the Internaiional Congress of Hisiochemistry and Cytochemistry, 4, 155. GAWLIK, Z, (1965) Morphological and morphochemical properties ofthe elastic system in the motor organ of man. Folia Hixiochemica and Cytochemica, 3, 233. GREENLEE, T . K . JR., ROSS, R. & HARTMAN, J . L . (1966) The fine structure of elastic hhzrs. Journal of Cell Biology, 30. 59PEARSE, A . G . E . (196*4) Histochemisiry: theoretical and applied, 3rd edn. Vol. i, p. 620. J. & A. Churchill Ltd., London. RODRIGO, F.G., COTTA-PEREIKA, G . SE RICHES, D.J. (1973) Ultrastructural observations on uninvolved skin in dermatitis herpetiformis. 'Elastic' versus 'reticular' fibrils. British Journal of Dermatology, 89, 543. Ross, R. & BORNSTEIN, P. (1969) The elastic fiber; the separation and partial characterization of its macromolecular components. Journal of Cell Biology, 40, 366.
Ultrastructural study of elaunin fibres in the secretory coil of human eccrine sweat glands G.COTTA-PEREIRA,* F.GUERRA RODRIGO AND S.BITTENCOURT-SAMPAIO Laboratory of Cell Biology, Gulbenkian Institute of Science, Ociras, Portugal; Department of Histology and Embryology, Faculty of Medicine, State University of Guanabara and Department of Histology and Embryology, Federal University of Rio dc Janeiro, Brazil: Department of Dermatology, Faculty of Medicine, Lisbon, Portugal Accepted for publication 7 March 1975
SUMMARY
The basement membrane of the secretory coil of human eccrine sweat glands was studied by light and electron microscopy. With classical staining methods for the elastic system, elaunin ring fibres were observed in the basement membrane ofthe secretory coil. The ultrastructural study showed that elaunin fibres are formed by bundles of fibrotubulcs 10-12 nm in diameter surrounding aggregates of amorphous material. These fibres differ from elastic fibres which are thicker and have an amorphous material more homogeneous and abundant.
In previous work, the distribution and morphology of the elastic system in the hixman dermis were studied and the existence of three types of fibres (oxytalan, elaunin and clastic fibres) was demonstrated (Bittencourt-Sampaio & Cotta-Pereira, 1971; Rodrigo, Cotta-Pereira & Riches, 1973; CottaPereira, Rodrigo & Bittencourt-Sampaio, 1975). The most superficial are the oxytalan fibres; these depart from the elaunin plexus, which is located parallel to the dermo-epidermal junction and is connected with the elastic fibres ofthe reticular dermis. The ultrastructure of elastic fibres is well known (Greenlee, Ross & Hartman, 1966; Ross & Bornstein, 1969). They consist of an amorphous substance identified as elastin surrounded by the 'elastic fibre microfibrils'. Recently, we observed that the oxytalan fibres are formed by bundles of fibrotubules similar to those visualized in elastic fibres, but devoid of elastin (Cotta-Pereira et al., 1975). The fine structure of elaimin fibres is not yet known. Observing sections of human dermis by light microscopy, we found elaunin fibres in the basement membranef ofthe secretory coil of human eccrine sweat glands (Cotta-Pereira & Bittencourt-Sampaio, * Reprint requests should be addressed to Dr Gerson Cotta-Pereira, Departamento de Histologia e Embriologia, Faculdade de Ciencias Mcdicas, Universidade do Estado de Guanabara, Avenida 28 de Setembro, 87, fundos. Vila Isabel. Estado de Guanabara, Ciid. Postal 20 000, Brazil. t In this paper the term 'basement membrane' is used to name the extraccliular support ofthe basal surface of epirhelia as observed by light microscopy. 'Basal lamina' is applied to the dense filamentous sheet visualized as a constituent of the basement membrane by electron microscopy. 623
624
G.Cotta-Pereira et al.
1973). The purpose of this investigation was to study this area with the electron microscope in order to describe the fine structure of the elaunin fibres. MATERIAL AND METHODS
Skin fragments biopsicd from different areas of human volunteers were fixed in 10",, formaldehyde and embedded in paraflin. Histological sections were stained by the following techniques: Vcrhoeff's iron haematoxylin, Weigert's resorcin fuchsin, Unna-Tanzer's orccin, and Gomori's aldehyde fuchsin. All these techniques were preceded or not by an oxidation with 40",, peracetic acid (Pearse, 1968). The PAS technique after treatment with diastase was also used associated with the Weigert's resorcin fuchsin.
FIGURE I. Elaunin fibres wrapping the secretory coil of a sweat gland. Weigcrt's resorcin fuchsin method (x 720).
Other fragments were fixed for 3 h in a mixture of tannic acid-glutaraldehyde (TAG), containing 0-25",, tannic acid and 3'\, glutaraldehyde in Millonig buffer (pH ^ 7-4) (Futaesaku, Mizuhira & Nakamura, 1972). After postfixation in 2",, osmium tetroxide they were embedded in Epon. The ultrathin sections were studied in a Siemens Elmiskop IA at 80 kV, after staining with uranyl acetate and lead citrate. RESULTS
By light microscopy, using resorcin fuchsin, orcein or aldehyde fuchsin, an envelope of fibres was observed surrounding the sweat gland secretory coil (Fig. i). When the resorcin fuchsin method was preceded by oxidation with peracctic acid, the fibrous structures were enhanced and thinner fibres intermingled with the thicker ones were visualized. After Verhoeff's stain, with or without previous oxidation, no fibres were observed (Fig. 2). The association PAS-resorcin fuchsin showed a double stained membrane surrounding the secretory coils. While the diastase-resistant PAS-positive inner layer formed a continuous envelope around the
FIGURE 2. Secretory coil of a swear gland. Elastic fibres are present only tar from the gland farrow). They arc not seen in the secretory coil basement membrane. Verhoeff's iron haematoxylin ( x 284}.
FIGURE 3. Basement membrane of a sweat gland secretory coil. Longitudinal section of an elaunin fibre, formed by patches of dense amorphous material (arrow) among fibrillar bundles (star). BL = basal lamina. AS = amorphous sheath ( x 32,400).
FIGURK 4. Basement membrane of a sweat gland secretory coil. Cross section of an elaunin fibre. Note the amorphous substance scattered among fibrotubular bundles ( x 32,400).
FIGURE 5. Basement membrane of a sweat gland secretory coil. Note the cross sectioned fibrotubules 10-12 nm in diameter (arrows) ( x 64,800).
Elaunin fibres in human sweat glands
627
FIGURE 6. Longitudinal section of an elastic fibre. The amorphous component is compact and homogeneous. Some microfibrils are observed in the periphery of the fibre ( x 16,200).
gland, the resorcin fuchsin stained outer layer showed different aspects. In cross sections, it often appeared as a discontinuous ring. In oblique or in longitudinal sections it was represented by a rank of points or parallel rods. The electron microscopic study of the secretory coil basement membrane showed that it is formed of a basal lamina connected on its inner face with the serous and myoepithelial cells by half-desmosomes, and on its outer face with an amorphous and low electron dense sheath. This amorphous sheath has an irregular thickness and it is surrounded by collagen fibrils and by elastic-like fibres formed by patches of an electron dense amorphous substance in the core of fibrotubular bundles (Figs 3-5). This amorphous material is not so compact and homogeneous as it is in the elastic fibres (Fig. 6). Bundles of fibrotubules 10-12 nm in diameter and without amorphous material were also visualized.
628
G.Cotta-Pereira et al, DISCUSSION
Previously, it was demonstrated by light microscopy that the classical staining methods for elastic tissue could reveal in the human dermis oxytaian, elaunin and elastic fibres (Bittencourt-Sampaio & Cotta-Pereira, 1971; Cotta-Pereira et al, 1975). Gawlik (1965) considered these three types of fibres as the normal constituents of the elastic system of connective tissues. In the present work parallel ring fibres wrapping the secretory coil of sweat glands were demonstrated by resorcin fuchsin, orcein or aldehyde fuchsin but not by the Verhoeff's method. Fibres with similar tinctorial characteristics were revealed in tendons, fibrous and hyaline cartilage and elastic laminae of developing aortae (Gawlik, 1965). They were named 'elaunin fibres' by Gawlik (1965). On the basis of these tinctorial affinities, the sweat gland ring fibres are elaunin fibres, differing from elastic fibres because they do not stain with Verhoeff's method. Using resorcin fuchsin after oxidation with peracetic acid, thinner fibres binding the thick ring fibres were revealed. They have therefore the same tinctorial characteristics as the oxytaian fibres (Fullmer & Lillie, 1958; Gawlik, 1965). It is concluded that a mesh-work of elaunin and oxytaian fibres involves the secretory coil of eccrine sweat glands. The main difficulty in studying elastic fibres with conventional electron microscopic techniques has been the irregular staining of their components. The introduction of tannic acid added to glutaraldehyde was a progress (Futaesaku ct al, 1972) simultaneously improving the fine details of the connective fibrils and giving a satisfactory staining of the elastin. With this method a good reproducibility of results is obtained. We tried several concentrations of tannic acid and the best results were observed with 0-25",, added to 3",, glutaraldehyde in Millonig's buffer. With this technique, the electron microscopic study of the ring fibres in cross and in longitudinal sections showed that they are formed by bundles of parallel fibrotubules 10-12 nm in diameter surrounding aggregates of amorphous material. They differ from elastic fibres which are thicker and where the amorphous material is more homogeneous and abundant. In his studies on elastogenesis, Gawlik (1965) found that in elastic laminae of developing aortae the oxytaian fibres appeared first and were later substituted by elaunin and finally by elastic fibres. Based on those observations oxytaian fibres may be considered as the most primitive elastic fibres. In agreement with this, are previous observations with the electron microscope demonstrating that oxytaian fibres are reduced to the fibrotubular component of the elastic fibres (Cotta-Pereira et al, 1975). In fact, during elastogenesis the fibrotubular component is the first to appear and thereafter the accumulation of amorphous material is progressive until the mature elastic fibre is formed (Fahrenbach, Sandberg & Cleary, 1966; Greenlee et al, 1966). In light microscopy, the concept of the elaunin fibre is only based on tinctorial data. As the ring fibres of sweat glands have elaunin characteristics we studied them with the electron microscope in order to investigate their fine structure. We found that they have ultrastructurai characteristics between the oxytaian and the elastic fibres. The fibrotubular bundles are abundant and the amorphous component is sparse. Functionally, elaunin and oxytaian fibres have been related to mechanical resistance because they are found in places where mechanical stress is exercised (Fullmer, 1958; Gawlik, 1965). Perhaps their mechanical resistance is based on the fibrotubular component. This hypothesis is in accordance with the absence of elastin in oxytaian fibres and its scarcity in elaunin fibres. The function of the elaunin ring fibres in the sweat coil could represent a mechanical support for the gland, completed by the thinner oxytaian fibres linking the thick elaunin ring fibres. Another fact to be discussed is the general structure of the basement membrane in the secretory coil of human eccrine sweat glands. From our observations, the inner layer, PAS-positive after
Elauninfibresin human sweat glands
629
diastase treatment, corresponds in electron microscopy to the area formed by the basal lamina and the amorphous sheath surrounding it. The outer layer is therefore constituted by elaunin, oxytalan and collagen fibres. ACKNOWLEDGMENTS Dr Cotta-Pereira was partly supported by the State Secretary of Science and Technology, Guanabara, Brazil. The authors wish to thank Dr J.F.David-Ferreira for his valuable discussion and criticism. Technical assistance was provided by Mr J.C.Lemos, Mrs Maria Teresa Plantier and Mrs Maria da Rcssurrei^ao Alpior^a. Miss Maria Manuela Magalhaes typed the manuscript.
REFERENCES BtTTENCOURT-SAMPAio, S. & COTTA-PEREIRA, G . (1971) Distribui^ao das libras elasticas, elauninicas e oxicalanicas na derme superior em pele humana. Anais Brasileiros de Dermaiologia, 46, 333. COTTA-PEREIRA, G . & BITTENCOURT-SAMPAIO, S. (1973) Contribui^ao ao estudo da cstrutura da membrana basal de glandulas sudoriparas ecrinas. Anais Brasileiros de Dermatalogia, 48, 319. COTTA-PEREIRA, G., RODRIGO, F . G . & BITTENCOURT-SAMPAIO, S. (1975) Oxytalan, elaunin and elasUc fibers in the human skin. Journal of Invesiigative Dermatology. Submitted for publication. FAHRENBACK, W.H., SANDBERG, L.B. & CLEARY, E . G . (1966) Ultrastruccural studies on early elastogenesis. Anatomical Record, 155, 563. FULLMER, H . M . (1958) Differential staining of connective tissue fibers in areas of stress. Science., 127, 1240. FULLMER, H.M. & LILLIE, R . D . (1958) The oxytalan fiber: a previously undescribed connective tissue fiber. Journal of Histochemistry arid Cyiochertiistry, 6, 425. FuTAESAKU, Y., MizuHiRA, V. & NAKAMURA, H . (1972) A new fixation method using tannic acid for electron microscopy and some observations of biological specimens. Proceedings of the Internaiional Congress of Hisiochemistry and Cytochemistry, 4, 155. GAWLIK, Z, (1965) Morphological and morphochemical properties ofthe elastic system in the motor organ of man. Folia Hixiochemica and Cytochemica, 3, 233. GREENLEE, T . K . JR., ROSS, R. & HARTMAN, J . L . (1966) The fine structure of elastic hhzrs. Journal of Cell Biology, 30. 59PEARSE, A . G . E . (196*4) Histochemisiry: theoretical and applied, 3rd edn. Vol. i, p. 620. J. & A. Churchill Ltd., London. RODRIGO, F.G., COTTA-PEREIKA, G . SE RICHES, D.J. (1973) Ultrastructural observations on uninvolved skin in dermatitis herpetiformis. 'Elastic' versus 'reticular' fibrils. British Journal of Dermatology, 89, 543. Ross, R. & BORNSTEIN, P. (1969) The elastic fiber; the separation and partial characterization of its macromolecular components. Journal of Cell Biology, 40, 366.
