A comparison of the immunofluorescent localization of collagen types I,III, and V with the distribution of reticular fibers on the same liver sections of the snow monkey (Macaca fuscata)

Summary Localizations of collagen types I, III, and V in monkey liver, as determined by the indirect immunofluorescence method, were photographically superimposed on the fibers revealed by silver-staining in the same tissue sections. Immunofluorescence for type I collagen was found to correspond with the brown collagen fibers and with some of the coarse reticular fibers, while that for type III collagen was found to correspond with most, but not all, reticular fibers of the liver as well as with the brown collagen fibers. The distribution of type V collagen coincides not only with the collagen fibers in the stroma of portal triads and around the central veins, but also with the coarse and fine reticular fibers in the liver lobules. By immuno-electron microscopy, reaction products with anti-type III and V collagens antibodies were demonstrated on cross-striated collagen fibrils, about 45 nm in diameter, in the space of Disse. From these observations, it is concluded that: (1) the fine reticular fibers are mainly composed of type III and type V collagens, and (2) the collagen fibers and coarse reticular fibers in the periphery of liver lobules are composed of type I, type III and type V collagens.     

Histochemical and morphological characterization of reticular fibers

The results presented in this paper show that collagen fibers can be clearly distinguished from reticular fibers using the picrosirius-polarization method. A morphologic and morphometric study of these two types of fibers with electron microscopy shows that reticular fibers are characterized by the smaller diameter of their fibrillar components and the higher content of interfibrillar material, resulting in a loose arrangement of the fibrils. The evidences presented suggest that the amorphous matrix in which fibrils are embedded is responsible for the silver impregnation of reticular fibers. Our results show that the matrix of reticular fibers is characteristically rich in heparitin sulfate, and that the glycosaminoglycans present show a high interaction with the fibrillar component of these fibers.     

Histochemical and morphological characterization of reticular fibers

Summary The results presented in this paper show that collagen fibers can be clearly distinguished from reticular fibers using the picrosirius-polarization method. A morphologic and morphometric study of these two types of fibers with electron microscopy shows that reticular fibers are characterized by the smaller diameter of their fibrillar components and the higher content of interfibrillar material, resulting in a loose arrangement of the fibrils. The evidences presented suggest that the amorphous matrix in which fibrils are embedded is responsible for the silver impregnation of reticular fibers. Our results show that the matrix of reticular fibers is characteristically rich in heparitin sulfate, and that the glycosaminoglycans present show a high interaction with the fibrillar component of these fibers.     

Production and characterization of a monoclonal antibody to human type III collagen

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.     

Collagen types I, III, and V in human embryonic and fetal skin

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.     

Collagen fibril diameters in arteries of mice. A comparison of manual and computer-aided morphometric analyses

Arteries of mice were studied by a silver impregnation technique, by the Picrosirius-polarization method and by transmission electron microscopy. The histochemical results obtained coincided with the electron-microscopic observations in showing the presence of two distinct collagen populations, segregated into different compartments of each artery. The fibrous component of the tunica media was comprised of reticulin fibers, which displayed a distinct argyrophilia when studied by means of the silver impregnation technique, and showed up as thin, weakly birefringent, greenish fibers when examined with the aid of the Picrosirius-polarization method. In addition, the electron-microscopic studies disclosed the presence of thin collagen fibrils in the tunica media, contrasting with the thicker fibrils that could be localized ultrastructurally to the tunica adventitia where nonargyrophil, coarse collagen fibers had been characterized by the histochemical methods used. In this respect, collagen distribution in arteries of mice is very similar to the pattern that was consistently observed in the other species studied, which argues in favor of the existence of a uniform structural pattern of collagen distribution that is a general phenomenon in vertebrate arteries. Experimental results comparing the traditional method and the computer-aided measurement of collagen fibril diameters showed that the system provides results equivalent to those produced by manual execution. In addition, the advantage in speed of the computer-aided method should prove useful in complicated studies where numerous structures are involved.     

Ultrastructural organization of the basic substance of human dermis]

The data on ultrastructural organization of the ground substance in the human dermis obtained electron histochemically are represented. Five types of ruthenium positive structures of polysaccharide origin are detected: retinal structure (I), amorfous substance (II), membranes of collagen fibrils (III) and elastic fibres (V), fine ruthenium positive streakness of collagen fibrils (IV). These structures, except fine streakness, form a united polysaccharide system of the dermis participating in maintenance of structural-functional integrity of the connective tissue (collagen-elastic) carcass of the dermis. Two mechanisms, interconnected and oppositely directed, perform this function: the buffer mechanism preventing the connective tissue fibers and collagen fibrils to approach each other, and the binding mechanism preventing the fibrils and fibers to dissociate. The reticular structure performs mainly this function at the level of fibers, and the amorphous substance does it at the level of fibrils.     

Type V collagen distribution in liver is reconstructed in coculture system of hepatocytes and stellate cells; the possible functions of type V collagen in liver under normal and pathological conditions

The contents of type I, type III and type V collagen and the collagen type specific distributions in liver under normal and cirrhotic conditions were examined. In CCl4 injected rat, the increasing amount of type V collagen was a specific event during the progression of cirrhosis. In normal liver, immunohistochemical observation showed that type V collagen was localized on the fine fibrils, while type I was localized on the thick fibril. Type V collagen was partially colocalized with type IV collagen. In the cirrhotic liver, type V collagen was localized on the margin of the thick fibrous septa along with type IV collagen. Type I collagen existed in the core region of fibrous septa where the stellate cells were prominent. To elucidate the mechanism of the type specific deposition of collagen in the liver, we constructed a coculture system using both stellate cells and hepatocytes. In this system, type V collagen was mainly deposited on hepatocyte colonies not on stellate cells, while type I collagen fibrils were localized on stellate cells. The spatial positioning of type I and type V collagens in vitro was similar to that in the liver. In the cell adhesion assay, the adhesion of stellate cells to type V collagen was poorer than that of the hepatocytes. The collagen type-specific affinity of the stellate cells and hepatocytes may explain the specific localization of type V collagen in the liver and coculture system. These results suggested that the functions of type V collagen are not only to connect type IV collagen with type I collagen fibril, but also to protect the parenchyma from excess type I collagen deposition produced by stellate cells under pathological conditions.     

Electron-microscopic localization of type II,IX, and V collagen in the organ of Corti of the gerbil

Summary The presence of types II, IX and V collagen was probed in the organ of Corti of the adult gerbil cochlea by use of immunocytochemistry at the light- and electron-microscopic levels. Type II collagen is found in the connective tissues of the osseous spiral lamina and spiral limbus. In the region of the sensory hair cells it is present in the tectorial membrane and antibodies bind to the thick unbranched radial fibers. Type IX collagen co-localizes with type II collagen in the tectorial membrane, where antibodies bind to the thick unbranched radial fibers. Type V collagen is present in the connective tissue of the spiral limbus, the osseous spiral lamina, the eighth nerve, and the tectorial membrane. In the tectorial membrane, the staining with antibodies to type V collagen is more diffuse than that seen for types II and IX collagen and antibodies to type V bind to the thin, highly branched fibers in which the thick fibers are embedded. The results indicate that collagens characteristic of cartilage are localized in the organ of Corti. Within the tectorial membrane, types II and IX collagen form heterotypic thick fibers embedded in a reticular network of type V collagen fibers. These collagens form a highly structured matrix which contributes to the rigidity of the tectorial membrane and allow it to withstand the physical stresses associated with transmission of the stimuli necessary for sensory transduction.     

Collagen type I and type V are present in the same fibril in the avian corneal stroma

The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Type VIII collagen has a restricted distribution in specialized extracellular matrices

A pepsin-resistant triple helical domain (chain 50,000 Mr) of type VIII collagen was isolated from bovine corneal Descemet's membrane and used as an immunogen for the production of mAbs. An antibody was selected for biochemical and tissue immunofluorescence studies which reacted both with Descemet's membrane and with type VIII collagen 50,000-Mr polypeptides by competition ELISA and immunoblotting. This antibody exhibited no crossreactivity with collagen types I-VI by competition ELISA. The mAb specifically precipitated a high molecular mass component of type VIII collagen (EC2, of chain 125,000 Mr) from the culture medium of subconfluent bovine corneal endothelial cells metabolically labeled for 24 h. In contrast, confluent cells in the presence of FCS and isotope for 7 d secreted a collagenous component of chain 60,000 Mr that did not react with the anti-type VIII collagen IgG. Type VIII collagen therefore appears to be synthesized as a discontinuous triple helical molecule with a predominant chain 125,000 Mr by subconfluent, proliferating cells in culture. Immunofluorescence studies with the mAb showed that type VIII collagen was deposited as fibrils in the extracellular matrix of corneal endothelial cells. In the fetal calf, type VIII collagen was absent from basement membranes and was found in a limited number of tissues. In addition to the linear staining pattern observed in the Descemet's membrane, type VIII collagen was found in highly fibrillar arrays in the ocular sclera, in the meninges surrounding brain, spinal cord, and optic nerve, and in periosteum and perichondrium. Fine fibrils were evident in the white matter of spinal cord, whereas a more generalized staining was apparent in the matrices of cartilage and bone. Despite attempts to unmask the epitope, type VIII collagen was not found in aorta, kidney, lung, liver, skin, and ligament. We conclude that this unusual collagen is a component of certain specialized extracellular matrices, several of which are derived from the neural crest.     

A Modified Silver Impregnation Technique for the Observation of Thick Sections of Lymph Nodes Perfused with Colloidal Carbon

For many years, a variant of the silver impregnation technique of Bielchowsky has been used to study the lymph node because it clearly outlines the various structures which are usually hard to contrast with standard staining methods. Like other variants of silver impregnation, this method blackens the cell nuclei as well as the reticular fibers; however, it inhibits the impregnation of the nuclear chromatin immediately adjacent to fibers. Hence, this variant selectively darkens the lymphoid cell populations of the nodal structures which contain a loose fiber network.

To study the blood vascular network of the lymph node based on perfusion of colloidal carbon, a staining procedure was needed which would contrast nodal structures on thick sections, while allowing the carbon-filled small blood vessels to be distinguished from the impregnated coarse reticular fibers. In an attempt to adapt this variant of Bielchowsky's technique, 10, 20, 40 and 60 nm thick sections from rat nodes, fixed in a solution of Bouin-Hollande for 72 hr, were silver impregnated with serial dilutions (1:2 to 1:128) of the ammoniacal silver solution. Forty-micrometer thick sections impregnated with a 1:16 dilution of the original silver solution at 37 C and for 30 min provided the best results for the conditions.     

Microwaves Applied to Silver Impregnations with Ammoniacal Silver Carbonate

Techniques for impregnation with ammoniacal silver carbonate provide valuable information on all types of tissue; however, the time investment required to impregnate a few sections has limited their application. We have shortened the impregnation times by using microwaves in techniques for reticular fibers, astrocytes, nerve fibers and chromaffin cells. The results were satisfactory with markedly reduced impregnation time and elimination of nonspecific silver deposits.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Reticular meshwork of the spleen in rats studied by electron microscopy

The reticular meshwork of the rat spleen, which consists of both fibrous and cellular reticula, was investigated by transmission electron microscopy. The fibrous reticulum of the splenic pulp is composed of reticular fibers and basement membranes of the sinuses. These reticular fibers and basement membranes are continuous with each other. The reticular fibers are enfolded by reticular cells and are composed of two basic elements: 1) peripheral basal laminae of the reticular cells, and 2) central connective tissue spaces in which microfibrils, collagenous fibrils, elastic fibers, and unmyelinated adrenergic nerve fibers are present. The basement membranes of the sinuses are sandwiched between reticular cells and sinus endothelial cells and are composed of lamina-densalike material, microfibrils, collagenous fibrils, and elastic fibers. The presence of these connective tissue fibrous components indicates that there are connective tissue spaces in these basement membranes. The basement membrane is divided into three parts: the basal lamina of the reticular cell, the connective tissue space, and the basal lamina of the sinus endothelial cell. When the connective tissue space is very small or absent, the two basal laminae may fuse to form a single, thick basement membrane of the splenic sinus wall. The fibrous reticulum having these structures is responsible for support (collagenous fibrils) and rebounding (elastic fibers). The cells of the cellular reticulum--reticular cells and their cytoplasmic processes, which possess abundant contractile microfilaments, dense bodies, hemidesmosomes, basal laminae, and a well-developed, rough-surfaced endoplasmic reticulum, and Golgi complexes, which are characteristic of both fibroblasts and smooth muscle cells--are considered to be myofibroblasts. They may play roles in splenic contraction and in fibrogenesis of the fibrous reticulum. The contractile ability may be influenced by the unmyelinated adrenergic nerve fibers that pass through the reticular fibers. The three-dimensional reticular meshwork of the spleen consists of sustentacular fibrous reticulum and contractile myofibroblastic cellular reticulum. This meshwork not only supports the organ but also contributes to a contractile mechanism in circulation regulation, in collaboration with major contractile elements in the capsulo-trabecular system.     

Extracellular matrix of different composition supports the various splenic compartments of guinea fowl (<Emphasis Type="Italic">Numida meleagris</Emphasis>)

The extracellular matrix (ECM) of the spleen of the guinea fowl has been studied by immunohistochemistry. Each splenic compartment contains a different composition of ECM. Reticular-fiber-specific collagen III is expressed by the red pulp and thymus-dependent periarterial lymphatic sheath, but silver impregnation reveals a reticular-fiber-like structure in the periellipsoidal white pulp (PWP) where collagen III is absent. The penicillar capillaries of one central artery are enveloped by a single branching sheath or sleeve: the ellipsoid or Schweigger-Seidel sheath. The shape of the sleeve shows more resemblance to a deer antler than to an ellipsoid; it emerges at the beginning of the penicillar capillaries and ends at the edge of the PWP. It is wrapped by a novel discontinuous basement-membrane-like structure that expresses tenascin and that is named the capsule of the Schweigger-Seidel sheath (CSS). The cuboidal-shaped inner cell layer of the ellipsoid can be identified by a novel monoclonal antibody: BID3. BID3-positive stellate-shaped cells also occur in the PWP, suggesting that this cell population has migratory capability. Monoclonal antibody KIF8 recognizes an ECM component in the ellipsoid not only of guinea fowl, but also of chicken and quail, and may thus identify an ellipsoid-specific antigen. Collagen I is associated with both the basement membrane of the penicillar capillary and the CSS, whereas collagen III is present only in the CSS. Laminin is expressed in the red pulp, but its staining pattern does not indicate the presence of the "ring fibers", which suggests the absence of sinuses. Fibronectin is the only ECM molecule studied that occurs in every splenic compartment, indicating extensive intrasplenic cell migration.     

Immunoelectron microscopic localization of collagens type I, V, VI and of procollagen type III in human periodontal ligament and cementum

We examined the ultrastructural localization of collagens Type I, V, VI and of procollagen Type III in decalcified and prefixed specimens of the periodontal ligament and cementum, by immunoelectron microscopy using ultra-thin cryostat sections. Immunostaining for collagen Type I was pronounced on the major cross-striated fibrils entering cementum and in cementum proper, whereas staining for procollagen Type III was almost exclusively observed on the major fibrils in the periodontal ligament situated more remote from cementum. Reactivity for collagen Type V was limited to aggregated, unbanded filamentous material of about 12 nm diameter that was found mainly in larger spaces between bundles of cross-striated collagen fibrils and occasionally on single microfibrils that apparently originated from the ends of the major collagen fibrils, which may support the concept of this collagen as a component of core fibrils. Collagen Type VI was present as microfilaments appearing to interconnect single cross-striated fibrils. In the densely packed fibril bundles of the periodontal ligament, no collagen type VI was detected. Neither Type V or Type VI collagen was observed in cementum.     

Production of fibronectin and collagen types I and III by chick embryo dermal cells cultured on extracellular matrix substrates

Dermal cells isolated from the back skin of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III, or IV collagen, fibronectin and several glycosaminoglycans (GAGs): hyaluronate, chondroitin-4, chondroitin-6, dermatan and heparan sulfates). The effect of these substrates on the production of fibronectin, of types I, III and IV collagen by cells was compared with that of culture dish polystyrene. Using immunofluorescent labeling of cultured cells, it was observed that, on all substrates, in 1-day and 7-day cultures, 85 to 95% of cells contain type I collagen in the perinuclear cytoplasm; label was absent from cell processes. Type I collagen was also detected in extracellular fibers extending between neighboring cells. By contrast, on all substrates, only 5 to 20% of cells produced type III collagen. Otherwise distribution of type III collagen was similar to that of type I collagen. With anti-type IV collagen antibody no staining of either cell content or extracellular spaces was detected. Staining with anti-fibronectin antibody revealed two types of distribution patterns. On polystyrene and on all but type I collagen substrates, labeling revealed clusters of short thick strands and patches of fibronectin-rich material in extracellular spaces. On type I collagen substrate, however, immunostaining revealed a delicate network of regularly spaced parallel fibrils of fibronectin extending between and along cells. Using quantitative radioimmunoassay of the culture media, it was shown that, after 7 days of culture, cells secreted more type I than type III collagen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microfibrils: a constitutive component of reticular fibers in the mouse lymph node

Summary Fibrous components other than collagen fibrils in the reticular fiber of mouse lymph node were studied by electron microscopy. Bundles of microfibrils not associated by elastin and single microfibrils dispersed among collagen fibrils were present. The diameter of the microfibrils was 13.29±2.43 nm (n=100). Elastin-associated microfibrils occurred at the periphery of the reticular fiber. Elastin was enclosed by microfibrils, thus forming the elastic fiber, which was clearly demonstrated by tannic acid-uranyl acetate staining. In the reticular fiber of lymph nodes, the elastic fiber consisted of many more microfibrils and a small amount of elastin. These microfibrils, together with the collagen fibrils, may contribut to the various functions of the reticular fibers.