Immunogold Labeling Method Research Articles

Met5‐enkephalin is localized within axon terminals in the subfornical organ: Vascular contacts and interactions with neurons containing gamma‐aminobutyric acid

Met5-enkephalin inhibits sodium and water excretion and antagonizes the central actions of angiotensin II in subfornical organ of rat brain. We examined the ultrastructural basis for enkephalin modulation in this circumventricular region. Additionally, we examined the possibility that there might be cellular sites for functional interactions involving Met5-enkephalin and gamma-aminobutyric acid (GABA), a known inhibitory transmitter throughout the central nervous system. Met5-enkephalin and GABA were identified in single coronal sections through the subfornical organ using immunoperoxidase and silver-enhanced immunogold labeling methods, respectively. Enkephalin-like immunoreactivity was most prominently localized within axon terminals. These were distributed primarily in the central, highly vascular, regions of the subfornical organ. Enkephalin-labeled terminals were apposed to the basement membranes of fenestrated capillaries and also formed symmetric, inhibitory type synapses with neurons. In terminals associated with either blood vessels or neurons, the enkephalin immunoreactivity was enriched in large (80-150 nm) dense core vesicles. The immunoreactive vesicles were usually located within portions of the axon in close proximity to astrocytic processes. In contrast, smaller vesicles in the same terminals were more often aggregated near the basement membrane of the capillaries and the active zone of the synapse. The targets of enkephalin-immunoreactive terminals were either unlabeled or GABA-labeled dendrites of local neurons. Enkephalin was also co-localized with GABA in perikarya and in axon terminals. Terminals containing only GABA were far more abundant than those containing enkephalin or enkephalin and GABA. GABA-immunoreactive terminals formed symmetric synapses on unlabeled dendrites some of which also received convergent input from terminals containing enkephalin. Additionally, the enkephalin-immunoreactive terminals were closely apposed to GABA-labeled and unlabeled terminals. These results suggest sites for nonsynaptic release of Met5-enkephalin from dense core vesicles in contact with astrocytes near blood vessels and synaptic complexes in the rat subfornical organ. Moreover, the observed dual localization and pre- and postsynaptic associations between neurons containing Met5-enkephalin and GABA indicate that inhibitory effects of opioids in the subfornical organ may be mediated or potentiated by GABA.

Distribution of alpha-galactosyl-containing epitopes on Trypanosoma cruzi trypomastigote and amastigote forms from infected Vero cells detected by Chagasic antibodies.

Reactivity of different Trypanosoma cruzi developmental forms with purified Chagasic anti-alpha-galactosyl antibodies (anti-Gal) was studied using epimastigotes from axenic cultures, trypomastigotes and amastigotes from infected Vero cell cultures, and an immunogold labeling method as observed by electron microscopy. Epimastigotes were poorly labeled, whereas extracellular trypomastigotes and amastigotes bound heterogeneously to the antibody with many cells being intensely labeled at the cell surface, including the membrane lining the cell body, the flagellum and the flagellar pocket. Parasites with poor labeling at the cell surface generally had several gold particles within the cell, mostly in cytoplasmic vacuoles. The Golgi complex of trypomastigotes was strongly labeled. Intracellular parasites were labeled at the parasite cell surface or within vacuolar structures. The expression in T. cruzi-infected Vero cells of alpha-galactosyl antigenic structures acquired from the parasite was shown by moderate labeling with Chagasic anti-Gal of the membrane lining parasite-free outward cell projections. The reactivity with purified anti-Gal from healthy individuals at the same concentrations of Chagasic anti-Gal was poor, with gold particles appearing in the nucleus and cytoplasm but not at the cell surface. It paralleled the labeling with Bandeireae simplicifolia IB-4 lectin. The results provide a basis for autoimmune reactions involving anti-Gal from chronic Chagasic patients.

Analysis of Immunolabeled Cells by Atomic Force Microscopy, Optical Microscopy, and Flow Cytometry

In this study we investigated the applicability of the (silver-enhanced) immunogold labeling method for atomic force microscopy. Human lymphocytes were labeled with anti-CD3 conjugated to fluorescein isothiocyanate and a secondary antibody (goat anti-mouse) linked with 1- or 30-nm colloidal gold particles. Silver enhancement was applied on these labeled cells to increase the size of the labels. In a setup combining an inverted optical microscope and a stand-alone atomic force microscope, a direct correlation was made between the force and the fluorescent images. Additionally, we performed flow cytometric analysis. From the results we conclude that immunogold labeling using small labels (1 nm) in combination with silver enhancement (30 min) proves to be a reliable method for high-resolution cell surface antigen detection in atomic force microscopy.

Multiple Labeling with Silver Enhanced Gold Probes

Abstract Irnmunogold-silver staining (IGSS) often improves the detection efficiency of probes for light microscopy and scanning and transmission electron microscopy studies. This technique is not often used in conjunction with double immunogold labeling at either the ultrastructural or light level as the very size differences used to differentiate the probes are obscured by the silver shells. However, it is possible to use an indirect two sided double immunogold labeling method with silver enhancement that does allow some labeling specificity. Each side of the nickel grid carrying the section is immunolabeled as a separate procedure, which includes the initial use of a blocking agent on each side to reduce nonspecific labeling. Although a single probe size can be used for both section faces, only one side of the grid is silver enhanced. The great depth of field of the transmission electron microscope permits the resolution of both probes in their respective specimen planes. A single probe size reduces the...

N-Acetyl-d-glucosamine-specific lectin purified fromVibrio cholerae01

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.

Light microscopic and ultrastructural distribution of type VI collagen in human liver: alterations in chronic biliary disease.

We have investigated the distribution of type VI collagen in normal human liver obtained from cadaveric renal transplant donors, using a peroxidase-antiperoxidase method for light microscopic visualization, and an immunogold labelling method for ultrastructural localization. The distribution was compared with that of the more abundant interstitial collagen type III, using antibodies to amino terminal procollagen type III. Staining for type VI collagen was identified in Glisson's capsule, in portal tract stroma and within the space of Disse. Perisinusoidal staining showed intra-acinar heterogeneity with the intensity in acinar zones 2 and 3 being greater than in zone 1. Type III collagen was also found in the space of Disse although no significant intra-acinar variation in staining intensity was noted. Immuno-gold labelling for type VI collagen was demonstrated on amorphous or microfilamentous material lying between, and occasionally appearing to interconnect, cross-striated collagen fibrils, whereas labelling for amino terminal procollagen type III was exclusively on fibrils. Intracellular staining for type VI collagen was noted in perisinusoidal (lto) cells. These results confirm that type VI collagen is a ubiquitous constituent of the normal hepatic extracellular matrix and suggest that it may be synthesized by perisinusoidal (lto) cells. The distribution of type VI collagen was also studied in biopsy material from patients with different histological stages of primary biliary cirrhosis. Intense staining was noted around proliferating bile ductules within developing fibrous septa and in established septa of cirrhotic liver. These observations indicate that this 'minor' matrix component may play an important role in hepatic fibrogenesis.

Antigenic localities in the tissues of Paragonimus westermani by developmental stages using immunogold labeling method

In order to observe the antigenic localization in the tissues of Paragonimus westermani of developmental stages, immunogold labeling method was applied using serum of the cats which were infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissues from each developmental stage were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the young adult worm tissue of 4 weeks after infection with metacercariae, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cells as well as the secretory granules in the parenchymal tissue. The antigenic materials in the adult worm tissue were specifically concentrated on the secretory granules in the parenchymal tissue, the cytoplasm between granules in the vitelline gland and the epithelial lamella in the lumen of the caecum.

Glycine-like immunoreactive input to sympathetic preganglionic neurons

The organization of glycine-like immunoreactive (GLY-LIR) processes was investigated within the sympathetic preganglionic neuropils of male Sprague-Dawley rats and pigeons ( Columba livia). Sympathetic preganglionic neurons were retrogradely labeled with horseradish peroxidase following injections into the superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. Glycine-like immunoreactivity was visualized using postembedding immunoperoxidase and immunogold labeling methods. The neuropils surrounding pigeon sympathetic preganglionic neurons in the principal preganglionic cell column (nucleus of Terni) and in the nucleus intercalatus contained numerous GLY-LIR puncta. Many of these processes appeared to be ‘terminal-like’ swellings which closely apposed retrogradely labeled preganglionic perikarya and proximal dendrites. GLY-LIR somal and dendritic processes were intermingled among retrogradely labeled preganglionic neurons in the nucleus of Terni. None of these GLY-LIR cells were retrogradely labeled. The neuropils surrounding sympathetic preganglionic neurons in the rat also contained numerous GLY-LIR puncta; there were, however, qualitative differences in the density of such profiles across the preganglionic subnuclei. Within the central autonomic and intercalated regions there were numerous GLY-LIR processes, many of which closely apposed retrogradely labeled sympathetic preganglionic somas and proximal dendrites. Within the principal preganglionic cell column, the nucleus intermediolateralis pars principalis (Il p), there were very few GLY-LIR ‘terminal-like’ swellings closely apposed to cell bodies in regions of high somal packing density. In regions were this density diminished, GLY-LIR puncta closely apposed retrogradely labeled perikarya and proximal dendritic processes. GLY-LIR spinal neurons were never observed to be within Il p proper but were present in areas immediately dorsal (lateral lamina V), medial and ventral (lateral lamina VII). GLY-LIR neurons were never retrogradely labeled. The ultrastructural features of GLY-LIR terminals within the sympathetic preganglionic neuropils of both vertebrates were nearly identical. GLY-LIR terminal boutons formed synaptic contacts with retrogradely labeled preganglionic somas as well as with large and medium-sized proximal dendrites. The majority of identified GLY-LIR terminals, however, contacted non-retrogradely labeled medium and small caliber dendrites within the preganglionic neuropils. Ninety-eight percent of GLY-LIR synapses formed symmetric specializations with the postsynaptic element. Ninety-six percent of the GLY-LIR terminal boutons contained some combination of pleomorphic vesicles. These light and electron microscopic observations support the hypothesis that glycine is localized in terminals presynaptic to sympathetic preganglionic perikarya and dendrites. This presynaptic localization strengthens existing physiological and pharmacological data and is consistent with the suggestion that glycine may be functioning as an inhibitory neurotransmitter in the sympathetic preganglionic neuropils of vertebrates.

Antigenic localities in the tissues of Metagonimus yokogawai in the period of growth

In order to observe the antigenic localization in the tissues of Metagonimus yokogawai in growth stages, immunogoldlabeling method was applied to using serum of the cat which infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissues from each growth stages were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the worm tissues of all experimental groups, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cell as well as the secretory granules in the parenchymal tissue. In the 16th and 20th week grown worm tissues, the gold particles were specifically concentrated on the vesicles in the tegumental syncytium and cytoplasm of the tegumental cell. The gold particles were specifically concentrated on the caecal epithelia of the 4th, 8th and 12th week growth groups but slightly concentrated on those of the 16th and 20th week.

C-terminal tail of β-adrenergic receptors: immunocytochemical localization within astrocytes and their relation to catecholaminergic neurons in N. tractus solitarii and area postrema

β-Adrenergic receptors (βAR) in the medial nuclei of tractus solitarii (m-NTS) and area postrema (AP) may bind to catecholamines released from neurons, whereas only the AP has fenestrated capillaries allowing access to circulating catecholamines. Since varied autonomic responses are seen following βAR activation of the dorsal vagal complex, including the m-NTS and AP, we hypothesized that there might be a cellular basis for varied responses to βAR stimulation that depends pn the differential access to circulating catecholamines. Therefore, we comparatively examined the ultrastructural localization of the βAR in relation to catecholaminergic neurons in these regions. An antibody directed against the C-terminal tail (amino acids 404–418) of hamster β-adrenergic receptor (βAR404) was used in this study. The localization of βAR404 was achieved by the avidin-biotin peroxidase complex (ABC) technique in combination with a pre-embed immunogold labeling method to localize tyrosine hydroxylase (TH), the catecholamine-synthesizing enzyme. Within m-NTS and at subpostremal border, labeling for βAR404 was evident along the intracellular surface of plasma membranes of small, apparently distal, astrocytic processes. Astrocytic processes with βAR404-immunoreactivity formed multiple, thin lamellae around TH-labeled and non-TH neuronal cell bodies and dendrites. βAR404-immunoreactive astrocytes also extended end-feet around blood vessels and surrounded groups of axon terminals that were directly juxtaposed to each other. Some, but not all, of these axons demonstrated TH-immunoreactivity. Fewer βAR404-immunoreactive astrocytes were detected in AP, regardless of their proximity to catecholaminergic processes or blood vessels. The present astrocytic localization of βAR404, together with the earlier, neuronal localization of βAR's third intracellular loop, suggest that the βAR may be substantially different between neurons and astrocytes. The regional difference in the prevalence of βAR404-immunoreactive astrocytes suggests that these receptive sites may either: (i) be preferentially activated by catecholamines released from terminals rather than circulating catecholamines; or (ii) be down-regulated in AP due to blood-born substances, such as catecholamines. The extensive localization of βAR in the border between m-NTS and AP also suggests that catecholaminergic activation of these astrocytes may dictate the degree of diffusion of catecholamines which are of neuronal or vascular origin. The specific localization of βAR404-immunoreactivity to the more distal portions of astrocytes suggests the possibility that astrocytes have restrictive distributions of βAR and that the β-adrenergic activation lead to morphological or chemical changes that are also localized to the distal portions of astrocytes. Additionally, the detection of βAR404 in astrocytes contacting non-TH-immunoreactive neurons suggests the possibility for catecholaminergic modulation of non-catecholaminergic neurons via the activation of astrocytes.

Immunocytochemical localization of a specific antigen in cell walls of Penicillium islandicum

Immunofluorescence and immunogold labelling methods at the light and electron microscope levels respectively, were employed in order to determine the site(s) of binding of a monoclonal antibody PI01, specific for Penicillium islandicum , and raised in a previous study to soluble surface molecules of that fungus. By immunofluorescence labelling the PI01 antibody was localized at the walls of the vegetative hyphae but was not present in sporulating hyphae. By immunogold labelling the PI01 antibody was observed to bind to the outer wall layers in young actively-growing hyphae and at the interface between the outer pigmented and inner translucent wall layers in older hyphae and conidiophores. In conidiophores, the PI01-antigen appeared to diminish towards the distal ends of the metulae and the antigen was completely absent from phialides and conidia. We postulate that the specific antigen is an exoenzyme, possibly an autolysin and emphasise the importance of ultrastructural localization of antigens over immunofluorescence labelling techniques.

Quantitative analysis and postsynaptic targets of GABA-immunoreactive boutons within the rat trigeminal motor nucleus

We have used the post-embedding immunogold labelling method using antibodies to γ-aminobutyric acid (GABA) to obtain quantitative data on the distribution, frequency, postsynaptic targets and ultrastructural characteristics of GABA-immunoreactive (GABA-IR) boutons in the trigeminal motor nucleus of rats. We have also combined this method with horseradish peroxidase tracing to obtain specific evidence for termination of some GABA-IR boutons onto identified jaw-elevator motoneurones. Twenty-eight percent of all synapses in the motor nucleus involved GABA-IR boutons. Seventy-three percent of the GABA-IR boutons formed axo-dendritic synapses, 13% axo-somatic synapses and 14% axo-axonic synapses. Ninety-three percent of GABA-IR boutons formed symmetrical synapses. Overall, 58% of all boutons contained only flattened vesicles, while 26% contained round vesicles and 16% a mixture of vesicle types. Measurements of bouton crops sectional area, apposition length, and active zone length were obtained from serial reconstructions of 15 GABA-IR boutons and 30 unlabelled boutons. In each case mean values for GABA-IR boutons were significantly smaller than those for nonlabelled boutons.

Immunogold labelling of human von Willebrand factor adsorbed to collagen

von Willebrand factor (vWF) mediates adhesion of platelets to the exposed subendothelium at sites of vascular injury. This function is expressed through binding of vWF to both collagen and receptors on the platelet membrane. We have developed a new method using immunogold staining and electron microscopy, permitting visualization of human vWF adsorbed to collagen fibrils. The electron micrographs revealed strings of gold beads reflecting the polymeric structure of vWF. Our data showed dramatic differences in the binding of vWF to collagens of different sources: high binding density was observed using a collagen preparation isolated from aortic tissue whereas colloidal gold was virtually absent from tendon collagen. Using the immunogold labelling method we demonstrated that high shear rate enhanced vWF binding to aortic collagen.

Antigenic localities in the tissues of the young adult worm of Paragonimus westermani using immunogold labeling method

In order to observe the antigenic localization in the tissues of the young adult Paragonimus westermani, immunogold labeling method was applied using serum immunoglobulins(IgG) of the dog which infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex (particle size; 12 nm). It was observed by electron microscopy at each tissues of the worm. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were predominantly labeling on the epithelial lamela and lumen of caecum. The above finding showed that antigenic materials in young adult worm tissue were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells.

Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.

Increased immunoreactivity of collagen types I, III and V, fibronectin and TGF-β in retinal vessels of rats with experimental autoimmune uveoretinitis

The immunoreactivity of types I, III and V collagen, fibronectin and transforming growth factor-beta (TGF-beta) was studied by an immunogold labeling method in retinal vessels of rats with experimental autoimmune uveoretinitis (EAU) induced by retinal S-antigen. The basal lamina of retinal capillaries in normal rats showed low immunoreactivities for the extracellular matrix components and TGF-beta. However, at the peak of inflammation (day 13-16 postimmunization), labeling of all three types of collagen and fibronectin increased considerably in the basal lamina of retinal vessels. TGF-beta immunoreactivity was detected mainly in the vascular endothelium and the pericytes. The results suggest that TGF-beta synthesized by the endothelial cells and pericytes may regulate the synthesis and composition of extracellular matrix components in the vascular basal lamina during the course of EAU development.

Immunogold localization of basal laminar heparan sulfate proteoglycan in rat brain and retinal capillaries

Heparan sulfate proteoglycan was localized in the basal lamina of the brain and retinal capillaries, using an antibody to the core protein of the proteoglycan and a postembedding immunogold labeling method at the ultrastructural level. Gold particles appeared to be randomly distributed in the basal lamina of both types of capillaries, in contrast to discrete ruthenium red-staining sites near the endothelial plasma membrane, as shown in previous studies of the retinal vessel. The present study not only agrees with previous biochemical and immunofluorescence studies, but also provides a more precise localization of the antigen. Furthermore, the method can be useful for quantitative studies.

Trypanosoma congolense:appearance and distribution of variable antigen types during metacyclic differentiationin vitro

Differentiation of epimastigotes and production of infective metacyclic forms of Trypanosoma congolense were examined in a culture system which enabled manipulation of the population density of insect forms. Scanning electron microscopy of cultures revealed the attachment sites of epimastigotes in detail, showing them to be attached as 'clusters' or 'bundles' and having associated fibrillar structures. Dividing epimastigotes were observed either within individual bundles or in association with two bundles. Metacyclic forms were detected by an immunofluorescence antibody test (IFAT) using metacyclic variable-antigen type (M-VAT) specific monoclonal antibodies, by day 7 after seeding cultures. Trypanosomes expressing M-VATs appeared singly in bundles, observed by both IFAT and an immunogold labelling method. Statistical analysis using Poisson calculations suggested that, in general, the distribution of metacyclics expressing individual M-VATs was random throughout cultures.

A study on the body fluid antigen of Clonorchis sinensis using immunogold labeling method

In order to observe the antigenic localization in the tissues of the adult Clonorchis sinensis, immunogold labeling method was applied using serum immunoglobulins (IgG) of either worm-infected rabbits (group I) or antigen-immunized rabbits (group II) (by the body fluid obtained from the adult worms). The electron micrographs of the sectioned worm tissue antigens, embedded in Lowicryl HM 20 medium and stained with protein A-gold complex (particle size: 12 nm), were compared between the group I and group II. The gold particles were observed in the interstitial matrix of the worm parenchyma, the epithelial lamellae of the cecum, and the cecal lumen both in group I and II. But the particles were in general more concentrated in group II. The gold particles were not observed on the basal lamina of the tegument or on vitelline glands in group I, while they were highly concentrated on those areas in group II. There were also differences in the antigenicity of interstitial matrix(reacted with group I IgG) and head part(reacted with group II IgG) of the sperm cells in the seminal receptacle. Conclusively, it is suggested that the substances comprising the basal lamina of the tegument or vitelline glands act as specific antigens reacting with antigen(body fluid) immunized rabbit IgG. On the other hand, the substances in the cecal lumen and cecal epithelial lamellae are thought to be the specific antigen that react with the worm-infected rabbit IgG.

Immunoelectron Microscopical Evidence that Type V Collagen is a Fibrillar Collagen: Importance for an Aggregating Capability of the Preparation for Reconstituting Banding Fibrils

Type V collagen has already been shown, in many immunohistochemical studies, to be widely distributed in connective tissues. Its supramolecular structure, however, has been unclear. We demonstrate that the major aggregates formed from type V collagen solution in vitro are fine fibrils with a D-periodic banding pattern. Further, by using the immunogold labeling method, we find that these fibrils react strongly with anti-type V collagen antibody. Electronmicroscopic examination showed three kinds of aggregate: fine fibrils with periodic banding pattern, fine fibrils without banding pattern, and non-fibrillar materials. Both striated and nonstriated fibrils, when incubated with rat polyclonal anti-human type V collagen IgG followed by incubation with 15 nm-gold conjugated goat anti-rat IgG, were labeled with colloidal gold. We conclude that type V can be classified as a fibrillar collagen. Also, from the present findings together with previous studies, we believe type V collagen may exist in vitro in various connective tissues as fine fibrils with a 67 nm-periodic banding pattern, by itself, or with type I or type III fibrillar collagen, being located between, and connecting the basal lamina and interstitial collagen fibers.