Scanning Immunoelectron Microscopy Research Articles

The expression pattern of MUC1 glycoforms and other biomarkers of endometrial receptivity in fertile and infertile women

Changes in the surface epithelium of the endometrium, characterized in part by alterations in cell-surface molecules, sex steroid receptors and the appearance of pinopodes, coincide with the window of endometrial receptivity in the menstrual cycle. This study was performed to evaluate the usefulness of hematoxylin and eosin staining, scanning and transmission microscopy, and MUC1 glycoform, sex steroid receptor, and interleukin receptor (type 1) expression as biomarkers of endometrial receptivity using carefully characterized clinical fertile and infertile groups of women. Using a combination of immunohistochemistry and scanning electron microscopy (SEM) called scanning immunoelectron microscopy (SIM), we confirmed that MUC1 mucin was not associated with the endometrial pinopodes, which have been linked with embryo adhesion. We also showed that failure of embryo implantation was associated with an abnormal endometrial expression of MUC1 mucin, and retention of nuclear progesterone receptor (PR) particularly in epithelial cells. Hematoxylin and eosin staining, transmission electron microscopy (TEM), SEM in isolation and immunohistochemistry for interleukin receptor were not shown to be useful markers. Progesterone-dependent regulation of MUC1 appears to be an important factor in determining endometrial receptivity.

Three-dimensional expression of adhesion molecules on the superficial synovial intima of LPS-induced arthritis of the mouse knee analyzed by immuno-SEM.

Three-dimensional microlocalization of adhesion molecules, i.e. ICAM-1 (intercellular adhesion molecule), VCAM-1 (vascular adhesion molecule), LFA-1 (lymphocyte function-associated antigen), Mac-1 (macrophage differentiation antigen) and VLA-4 (very late activation antigen), expressed on type-A synoviocyte (macrophage-like cell) and type-B synoviocyte (fibroblast-like cell), were detected by immuno-scanning electron microscopy (SEM) to investigate the immunoreactive microenvironment of the superficial synovial intima in lipopolysaccharide (LPS)-induced arthritis of the mouse knee. Type-B synoviocytes extended rich slender processes from the periphery and constructed a cytoplasmic network, to which ICAM-1 was restricted. VCAM-1 was expressed only in the LPS-stimulated group and was relatively limited to the microvilli of type-B synoviocytes. Type-A synoviocytes were located randomly among the network with a smoother surface and expressed Mac-1 and LFA-1, which were counter-receptors for ICAM-1, and VLA-4 for VCAM-1 on the microvilli or lamellipodia. Three-dimensional microlocalization of adhesion molecules suggests that the network constructed by cytoplasmic processes and microvilli of type-B synoviocytes forms the pathway for the migration or the foothold for the fixation of type-A synoviocytes and takes part in forming an immunoreactive environment in the articular cavity.

Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells.

The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Morphological analysis of leucocyte transmigration in the pleural cavity.

The role that pleural mesothelial cells play in leucocyte transmigration into the pleural cavity was investigated in lipopolysaccharide-stimulated mice. Changes in mesothelial cell morphology and changes in expression of adhesion molecules on mesothelial cells and leucocytes were analysed by light microscopy, immunohistochemistry, transmission electron microscopy (TEM) and immuno-scanning electron microscopy (immuno-SEM). After stimulation, the mesothelial cells separated completely from one another before leucocyte penetration across the mesothelial layer occurred. These changes occurred primarily in the immediate vicinity of ribs, where a large number of leucocytes accumulated. Immuno-SEM showed that the expression of intercellular adhesion molecule-1 (ICAM-1) on the parietal pleural mesothelial cells was significantly up-regulated by lipopolysaccharide stimulation, and that of vascular cell adhesion molecule-1 (VCAM-1) was induced. Both were restricted to the microvilli of the mesothelial cells. By contrast, expression of intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1), mucosal addressin cell adhesion molecule-1 (MAdCAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1), peripheral node addressin (PNAd) and fibronectin were not detected. Lymphocyte function associated antigen-1 (LFA-1), macrophage-1 molecule (Mac-1) and very late appearing antigen-4 (VLA-4), all ligands of ICAM-1 and VCAM-1, were present on the transmigrated neutrophils and macrophages. These findings demonstrate that the immediate vicinity of ribs is a source of leucocyte migration into the pleural space.

Scanning electron microscopic observations of the basement membranes with dithiothreitol separation.

Scanning electron microscopic (SEM) studies of the interstitial surface of the lamina densa can be performed with dithiothreitol separation, which is the only method of exposing this surface. SEM observation revealed the three-dimensional structures of the meshwork in the lamina densa and anchoring fibrils in dithiothreitol-separated specimens. Detection of the components of the basement membrane can be performed by immunoscanning electron microscopy on this exposed surface by comparing the backscattered and the secondary electron images. SEM observation also revealed the fine structure of the lamina fibroreticularis using separated dermis or the lamina propria mucosae.

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage.

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.

Co-expression of Parathyroid Hormone and Chromogranin Ain Secondary Hyperparathyroidism: A Functional Marker for Secretory Activity of Hyperplastic Nodules

The relationship between secretion of parathyroid hormone (PTH) and biologic characteristics, including cell proliferation or monoclonality, is not yet fully understood. To evaluate secretory activity of glands or nodules histopathologically, we focused on the co-expression of chromogranin A (CgA) and parathyroid hormone (PTH) in each gland or nodule. A total of 55 glands from 38 patients with normal parathyroid glands, hyperplastic glands (diffuse and nodular) and primary adenomas were compared. Co-expression of PTH and CgA was decreased to 44.4% in diffuse hyperplastic glands, and to 39.6% in 91 hyperplastic nodules, in contrast to normal glands and primary adenomas that showed constant co-expression of PTH and CgA. Immunohistochemical study of PTH showed a coarse granular pattern predominantly in PTH-positive/CgA-positive nodules, and a dot-like pattern mainly in PTH-positive/CgA-negative nodules. Laser scanning microscopy and immunoelectron microscopy confirmed that a dot-like pattern is based on a positive reaction of PTH at the Golgi apparatus. MIB-1 LI was 12.6 +/- 11.6 in PTH-positive/CgA-positive, and 19.3 +/- 27.3 in PTH-positive/CgA-negative nodules. In conclusion, a combination of PTH and CgA could provide more information about the physiologic state of secretory activity of each nodule than does the simple observation of PTH immunoreactivity.

Analysis of Epitopes on Endometrial Epithelium by Scanning Immunoelectron Microscopy

Scanning immunoelectron microscopy was applied to human endometrial epithelium for the first time to simultaneously determine epitope localisation and cellular architecture. The method was established using HMFG1, an antibody to a glycoform of the MUC1 mucin. This was chosen because of the potential importance of MUC1 in connection with endometrial receptivity. Biopsies of mid-secretory phase endometrium were labelled using HMFG1 and silver-enhanced, gold-conjugated secondary antibody was then visualised by back-scattered electron imaging. The method provided a highly specific localisation of the HMFG1 epitope to the ciliated and ‘ciliogenic’ cells of the endometrial surface. In contrast, no reactivity was evident on the microvillous cells and endometrial pinopodes. The potential to integrate the study of the molecular and ultrastructural changes that occur in the endometrium by using scanning immunoelectron microscopy offers a powerful means of expanding our understanding of the adaptation of the endometrium in preparation for embryo implantation.

Three-Dimensional Morphological Analysis of Antigen-Antibody Reaction in Hepatic Sinusoids Preserved in Hypothermic UW Solution

ICAM-1 antigen-antibody reaction was visualized by three-dimensional immunoscanning electron microscopy of hepatic sinusoids in rat liver treated with hypothermic University of Wisconsin (UW) organ preservation solution. The results were compared with similar antigen-antibody reactions carried out with immunoliposomes injected in vivo. Morphologically, the hepatic sinusoids were preserved well during the hypothermic procedure. Endothelial cells had a large number of fenestrations, which partly aggregated and formed sieve plates. ICAM-1 expression was induced by injection of LPS and detected by monoclonal antibody in the UW solution followed by gold-labeled secondary antibody. ICAM-1 was restricted mostly to the unique areas of sieve plates with immature, small fenestrations. A similar distribution of ICAM-1 was present when detected by in vivo injection of immunoliposomes containing the monoclonal ICAM-1 antibody. The results showed that antigen-antibody reactions can take place in livers preserved in hypothermic UW solution. Further, the reaction is similar to that which could occur in vivo during transplantation. This suggests that it may be possible to block potentially harmful antigen-antibody reactions by addition of appropriate antibodies to hypothermic UW solution prior to transplantation.

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S1), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S2), fibrils were thicker than those found in S1 forming fibers and were densely deposited. The S2 layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.

Active and passive immunity against Borrelia burgdorferi decorin binding protein A (DbpA) protects against infection.

Borrelia burgdorferi, the spirochete that causes Lyme disease, binds decorin, a collagen-associated extracellular matrix proteoglycan found in the skin (the site of entry for the spirochete) and in many other tissues. Two borrelial adhesins that recognize this proteoglycan, decorin binding proteins A and B (DbpA and DbpB, respectively), have recently been identified. Infection of mice by low-dose B. burgdorferi challenge elicited antibodies against DbpA and DbpB that were sustained at high levels, suggesting that these antigens are expressed in vivo. Scanning immunoelectron microscopy showed that DbpA was surface accessible on intact borreliae. Passive administration of DbpA antiserum protected mice from infection following challenge with heterologous B. burgdorferi sensu stricto isolates, even when serum administration was delayed for up to 4 days after challenge. DbpA is the first antigen target identified that is capable of mediating immune resolution of early, localized B. burgdorferi infections. DbpA immunization also protected mice from B. burgdorferi challenge; DbpB immunization was much less effective. DbpA antiserum inhibited in vitro growth of many B. burgdorferi sensu lato isolates of diverse geographic, phylogenetic, and clinical origins. In combination, these findings support a role for DbpA in the immunoprophylaxis of Lyme disease and suggest that DbpA vaccines have the potential to eliminate early-stage B. burgdorferi infections.

Three-Dimensional Distribution of Intercellular Adhesion Molecule-1 on Lymphocytes in the High Endothelial Venule Analyzed by Backscatter Electron Imaging

The intercellular adhesion molecule-1 (ICAM-1) is a ligand for the β2 integrin. It is present on the vascular endothelium and plays an important role in cell to cell interaction in lymphocyte recirculation. ICAM-1 is expressed not only on the endothelium but also on other cells, especially lymphocytes adhering to the endothelium, B cells and mitogen-stimulated T cells. To define the functional role of ICAM-1 in lymphocytes, the three-dimensional distribution of ICAM-1 on the surface of lymphocytes in the rat mesenteric lymph node HEV was investigated by immunoscanning electron microscopy. ICAM-1 expression was closely associated with variations in surface structures of lymphocytes, which showed a cap-like structure, membrane ruffles and slender microvilli. ICAM-1 was preferentially localized on the cell surface including membrane ruffles (about 90% of all counted gold particles), but rarely on the microvilli (10%). The results suggest that activated lymphocytes with variations in surface structures demonstrate moderate expression and preferential localization of ICAM-1. This seems to enable a three-dimensional interaction of the microvilli of one lymphocyte with the surface membrane of another cell and might be a prerequisite for cluster formation between them, as seen in antigen presentation.

Immuno-scanning electron microscope characterization of large tubules in human deciduous dentin.

This study was undertaken to elucidate the type and origin of collagen fibrils which construct the large tubules in deciduous coronal dentin by scanning electron microscope and anti-types I and III collagen antibody procedures. The studies were performed on human deciduous teeth. The teeth were fixed in 4% paraformaldehyde solution and then fractured either mesio-distally parallel to the long axis of the tooth or transversely perpendicular to the long axis of the tooth crown. The specimens were three-dimensionally observed employing the scanning electron microscope to distinguish the content of large tubules. Polyclonal antibodies of anti-type I and anti-type III collagen with 20 nm colloidal gold, and secondary electron imaging and backscatter electron imaging of high-resolution field emission scanning electron microscopy were used to examine the types of collagen fibrils. The large tubules extended from the vicinity of the incisal edge of the dentino-enamel junction to the pulp cavity. Inside the large tubules, fibers in compact bundles run parallel to the longitudinal axis of the tubules. The fiber bundles consisted of collagen fibrils which were 50-150 nm in diameter with typical cross striation. Immuno-scanning electron microscopy showed type I collagen-labelling gold particles and type III collagen-labelling gold particles to be abundant on the fibrils. Types I and III collagen-labelling gold particles were present on the banded collagen fibrils regardless of their diameter. It was found that type III collagen is present together with type I collagen on the fibrils constructing the large tubules of the human deciduous dentin. This immunohistochemical study suggested that the fibrils constructing the large tubules were derived from the von Korff fibers, and types I and III collagens formed copolymers.

Temporal and spatial patterns of phosphotyrosine immunolocalization during cardiac myofibrillogenesis of the chicken embryo.

To investigate the possible role of phosphorylation of protein tyrosine during myofibrillogenesis (6- to 13-somite stages) of the chicken embryonic heart tube, immunolocalization of phosphotyrosine (P-Tyr) and the relationship between P-Tyr and developing myofibrils were studied by means of confocal scanning laser microscopy and immuno-electron microscopy. The staining pattern of P-Tyr varied in different sites of myocytes at different stages of embryonic development: At the cell-cell boundaries, P-Tyr was localized at the adhesion belt of outer myocardial layer cells (6- to 13-somite stages), non-junctional cell-cell contacts (6- to 13-somite stages) and early intercalated disks of both the outer and inner myocardial layer cells (8- to 13-somite stages). At the cell-extracellular matrix boundaries of inner layer cells, the first stages of myofibril formation appeared as serially aligned areas of P-Tyr localization closely associated with circumferentially aligned thick actin bundles (8- to 9-somite stages). This P-Tyr immunostaining decreased when the thick actin bundles developed into mature striated myofibrils at the 10- to 13-somite stages. These findings suggest that the phosphorylation of protein tyrosine residues is primarily concentrated at the modulating cell-cell and cell-matrix adhesion sites of developing myocytes and myofibrils.

High-resolution immuno-scanning electron microscopy using a non-coating method: study of herpes simplex virus glycoproteins on the surface of virus particles and infected cells.

The expression of two glycoproteins, i.e. glycoprotein C (gC) and glycoprotein D (gD), of herpes simplex virus type 1 (HSV-1) on the surface of extracellular particles of this virus was examined by immuno-scanning electron microscopy. Scanning electron microscopy specimens of infected cells immuno-labelled against the glycoproteins with colloidal gold particles were prepared by a conventional coating and a non-coating method. Surface ultrastructure of infected cells and gold particles were observed more clearly with specimens prepared by the non-coating method. The appearance of virus particles in association with glycoprotein expression on these particles and on the surface of infected cells was then studied. Progeny virus particles began to appear 6 h after infection, increased in number as the infection proceeded, and covered most of the cell surface by 16 h. Six to 24 h after the infection, the labelling density for each glycoprotein on virus particles remained constant. The labelling density for gD was always higher than that for gC. The patch-like distribution of gold-labelling against gD was often detected on infected cell monolayers at the exponential and late stage of one cycle of virus growth. The labelling density for gD on virus particles was the highest on these produced in Vero and L-929 cells, moderate in MRC-5, BHK-21 and FL cells, and the lowest in HEp-2 cells.

Immunological detection of the GerA spore germination proteins in the spore integuments of Bacillus subtilis using scanning electron microscopy

To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis, the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, l-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies. Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules. The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles. The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.

Platelet-Derived Microparticles Associate With Fibrin During Thrombosis

Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P-selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P-selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.

Immunological detection of the GerA spore germination proteins in the spore integuments ofBacillus subtilisusing scanning electron microscopy

To clarify the molecular mechanisms that trigger spore germination of Bacillus subtilis, the location of GerA proteins (GerAA, GerAB and GerAC), which were reported to be putative gene products of a receptor for one of the germinants, L-alanine, was investigated by immunological techniques using anti-GerA peptide antibodies. Four antibodies were raised against the corresponding epitopes, two in GerAA, one in GerAB and the other in GerAC molecules. The binding of all four antibodies to the inner surface of the cortex-less spore coat fragments could be seen by scanning immunoelectron microscopy with colloidal gold particles. The result agreed with the fact, previously reported, that the colloidal gold particles were visualized just inside the spore coat layer by transmission immunoelectron microscopy using another anti-GerAB peptide antibody.

Ultrastructural localization of the intercellular adhesion molecule (ICAM-1) on the cell surface of high endothelial venules in lymph nodes.

The high endothelial venules (HEV) in the lymph nodes are essential for lymphocyte recirculation. As a first step, the HEV surface interacts with lymphocytes through adhesion molecules. It is important to know where adhesion molecules are expressed on the surface ultrastructure and how these structures interact with lymphocytes. To demonstrate the ultrastructural mechanism of interaction between the HEV surface and lymphocytes through the intercellular adhesion molecule (ICAM-1), rat mesenteric lymph nodes were perfused through the superior mesenteric artery with the primary antibody (antirat ICAM-1 antibody) and secondary antibody (antimouse IgG coupled to 15 nm gold particles), which were diluted with hypothermic University of Wisconsin (UW) solution. After the immunoreaction, we analyzed the HEV three-dimensionally and quantitatively using immunoscanning electron microscopy (ISEM) combined with transmission electron microscopy (TEM). HEV expressed ICAM-1 in a 5-30-fold higher concentration than other vessels. Its distribution was extensive over the luminal surface of the cell down to the junctional area. The endothelial surface of HEV undulated to form branched microfolds, along which ICAM-1 was expressed. Cytoplasmic processes of lymphocytes were seen in microfurrows between microfolds and adhered to the sides of the folds. These observations imply that the microfolds expressing ICAM-1 and microfurrows are specific ultrastructural features for trapping lymphocytes, thus initiating lymphocyte emigration into the lymph node parenchyma.

Ultrastructural localization of the intercellular adhesion molecule (ICAM‐1) on the cell surface of high endothelial venules in lymph nodes

Background The high endothelial venules (HEV) in the lymph nodes are essential for lymphocyte recirculation. As a first step, the HEV surface interacts with lymphocytes through adhesion molecules. It is important to know where adhesion molecules are expressed on the surface ultrastructure and how these structures interact with lymphocytes. Methods To demonstrate the ultrastructural mechanism of interaction between the HEV surface and lymphocytes through the intercellular adhesion molecule (ICAM-1), rat mesenteric lymph nodes were perfused through the superior mesenteric artery with the primary antibody (antirat ICAM-1 antibody) and secondary antibody (antimouse IgG coupled to 15 nm gold particles), which were diluted with hypothermic University of Wisconsin (UW) solution. After the immunoreaction, we analyzed the HEV three-dimensionally and quantitatively using immunoscanning electron microscopy (ISEM) combined with transmission electron microscopy (TEM). Results HEV expressed ICAM-1 in a 5–30-fold higher concentration than other vessels. Its distribution was extensive over the luminal surface of the cell down to the junctional area. The endothelial surface of HEV undulated to form branched microfolds, along which ICAM-1 was expressed. Cytoplasmic processes of lymphocytes were seen in microfurrows between microfolds and adhered to the sides of the folds. Conclusions These observations imply that the microfolds expressing ICAM-1 and microfurrows are specific ultrastructural features for trapping lymphocytes, thus initiating lymphocyte emigration into the lymph node parenchyma. © 1996 Wiley-Liss, Inc.