Pre-embedding Immunoelectron Microscopy Research Articles

Structural Variations in Anchoring Fibrils in Dystrophic Epidermolysis Bullosa: Correlation with Type VII Collagen Expression

Dystrophic epidermolysis bullosa is characterized by various abnormalities of anchoring fibrils, which are mainly composed of type VII collagen, at the dermal-epidermal junction. To define these changes more clearly, we examined skin samples from 22 patients with different forms of dystrophic epidermolysis bullosa by pre-embedding immunoelectron microscopy using an antibody (LH 7:2) that binds to the NC-1 globular domain of type VII collagen, followed by 1 nm colloidal gold-labeled secondary antibodies and subsequent silver enhancement. In dominant dystrophic epidermolysis bullosa cases, there was only a slight but variable reduction in the immunolabeling density on anchoring fibrils and on the lamina densa, in parts similar to normal human skin. In localized recessive dystrophic epidermolysis bullosa skin, some fibrillar structures just below the lamina densa (and particularly subjacent to hemidesmosomes) had specific antibody labeling despite their lack of resemblance to definitive anchoring fibrils. Immunolabeling with LH 7:2 was also seen within basal keratinocyte endoplasmic reticulum and cytoplasmic vesicles in some dystrophic epidermolysis bullosa patients, usually with milder phenotypic features. Even in the most severe cases of generalized recessive dystrophic epidermolysis bullosa, occasional immunolabeling was found within the lamina densa and on scanty thin filamentous structures at sub-lamina densa sites usually occupied by anchoring fibrils. This study suggests that dystrophic epidermolysis bullosa patients express some type VII collagen NC-1 domain epitopes that may be variably reduced at the dermal-epidermal junction or retained within basal keratinocytes. The clinical heterogeneity in dystrophic epidermolysis bullosa is mirrored by a range of immunoelectron microscopy findings, indicating variability in completeness of anchoring fibril formation and a possible spectrum of underlying type VII collagen structural protein abnormalities.

Effect of fixation conditions on the granule morphology of rat antral gastrin cells: an ultrastructural and immunocytochemical study.

After fixation at pH = 7 or pH = 5 the gastrin (G) cells in the rat pyloric antrum were investigated by conventional and immunoelectron microscopy. After fixation at pH = 7 G cells contained numerous electron-lucent granules, a few electron-dense and intermediate granules while G cells had numerous electron-dense and intermediate granules, and a few electron-lucent granules after fixation at pH = 5. Preembedding immunoelectron microscopy revealed that in G cells after fixation at pH = 7, gastrin immunoreactivity was mainly seen in the cytoplasm, cores of a few electron-dense and intermediate granules and in the periphery but not within the numerous electron-lucent granules; in G cells after fixation at pH = 5, gastrin immunoreactivity was mainly visible in the cores of numerous intermediate and electron-dense granules, in the periphery of a few electron-lucent granules, but weakly in the cytoplasm. Therefore, the variable electron density of G cells may reflect differences in the degree of the leakage of contents from the gastrum-containing granules into the cytoplasm during fixation. This phenomenon may be related to the acidity of the granule substances during intragranular maturation.

Fodrin is a constituent of the cortical lattice in outer hair cells of the guinea pig cochlea: Immunocytochemical evidence

Localization of fodrin, a membrane skeletal protein, in the outer hair cell of the guinea pig cochlea was examined by immunocytochemical techniques. By immunofluorescence microscopy, fodrin was observed in the cuticular plate, in the infracuticular network and along the lateral wall. By immunoelectron microscopy of ultrathin cryosections, labeling for fodrin along the lateral wall was localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, pre-embedding immunoelectron microscopy of permeabilized specimens showed that most immunogolds for fodrin were on the thin cross-linking component of the cortical lattice. The results indicate that fodrin is a constituent of the cortical lattice which is thought to play an important role in outer hair cell motility.

Fine structure and possible origins of nerve fibers with corticotropin-releasing factor-like immunoreactivity in the rat central amygdaloid nucleus

The fine structure of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the central amygdaloid nucleus and CRF-containing afferents to the nucleus were investigated by pre-embedding immunoelectron microscopy and by the combination of fluoro-gold tracing and the indirect immunofluorescence method. Significant numbers of CRF nerve endings and dendrites formed synapses with non-immunoreactive dendrites and axon terminals, respectively. Axon terminals devoid of CRF frequently made synapses with the soma of immunoreactive and non-immunoreactive neurons; CRF nerve endings in contact with the soma were fewer in number. Occasionally, CRF was localized to both pre- and postsynaptic structures in the central amygdaloid nucleus. After flouro-gold injection into the central amygdaloid nucleus and adjacent areas, double-labeled cells with the tracer and CRF were observed mainly in the lateral hypothalamic area and occasionally in the dorsal raphe nucleus, and they were less numerous than single-labeled cells. These findings suggest that part of the CRF axon terminals identified in the electron micrographs arises from neurons in the lateral hypothalamic area and the dorsal raphe nucleus and the others from intra-amygdaloid CRF neurons. The immunoreactive dendrites are likely to derive from neurons in the central amygdaloid nucleus, which are shown to send axons to the lower brainstem. Thus, this study demonstrates that CRF structures constitute a more complex neuronal network in the central amygdaloid nucleus than previously considered.

Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.

This study represents a systematic analysis of the distribution of collagen types in human atherosclerotic lesions. Formalin-fixed, paraffin-embedded aortic tissues of 40 lesions from 16 different individuals ranging in age from 1 month to 84 years were examined immunohistochemically using antibodies to type I, III, IV, V, and VI collagens. Preembedding immunoelectron microscopy was used to simultaneously localize type V and VI collagens within the lesions. Localization of type III collagen was very similar to that of type I, and type VI collagen appeared together with these two types of collagen in the thickened intimas of all stages of the lesion. Type V collagen was not detected in either fatty streaks or the mild intimal thickening of the aortas of children. With advancing age and lesion progression, the immunoreactivity with anti-type V collagen antibody became more intense. Type IV collagen was detected in the basement membrane region of intimal cells. In advanced lesions thick deposits of type IV collagen were found around the elongated smooth muscle cells. Using immunoelectron microscopy, type V collagen was found to be localized to cross-banded collagen fibers, and type VI collagen was found to be localized to beaded filaments present throughout the interstitium of the thickened intima. These findings suggest that collagens preserve the pathophysiological and functional integrity of the vascular wall by providing mechanical support as well as assuring the proper interaction of cells during the formation of atherosclerotic lesions.

Cytokine Regulation of Proliferation and ICAM-1 Expression of Human Dermal Microvascular Endothelial Cells In Vitro

The effects of recombinant human interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) on the cell proliferation and the expression of intercellular adhesion molecule-1 (ICAM-1) were assessed in cultured human dermal microvascular endothelial cells (HDMEC). IL-1 alpha and IL-1 beta stimulated the proliferation of HDMEC in a dose-dependent manner, whereas in control experiments using human umbilical vein endothelial cells (HUVEC), IL-1 alpha and IL-1 beta did not stimulate HUVEC growth. Also GM-CSF stimulated the proliferation of HDMEC, whereas IL-6 did not affect endothelial cell growth in vitro. Treatment with IL-1 alpha, IL-1 beta, and TNF markedly increased the expression of ICAM-1 on HDMEC in a time- and dose-dependent manner, in contrast to IL-6 and GM-CSF. By pre-embedding immunoelectron microscopy, membrane-bound expression of ICAM-1 was visualized with pronounced labeling in areas of microvillous cell protrusions. The TNF-induced expression of ICAM-1 on HDMEC was blocked by co-incubation with a neutralizing antibody against TNF, but not with neutralizing antibodies against IL-1 alpha, IL-1 beta, or IL-6. In addition, co-incubation of HDMEC with TNF and the retinoid compound acitretin, dexamethasone, or indomethacin did not abrogate the TNF-induced ICAM-1 expression. These results disclose IL-1 as a major, multifunctional endothelial cell-targeted cytokine and further confirm the concept that pro-inflammatory cytokines exert differential regulatory effects on dermal microvascular endothelial cell proliferation and expression of cell-adhesion molecules.

Use of LR white resin for post-embedding immunolabelling of brain tissue.

The object of this study was to investigate the applicability of the acrylic resin 'LR White' to immunolabelling of various antigenic determinants in aldehydefixed rat CNS tissue. Antibodies were used, which worked well in paraffin sections and therefore were suitable to detect antigens resistant to complete dehydration and heat. Different LR White embedding protocols were employed in order to select the preparation conditions that adequately preserved both the antigenicity and fine structure. Specimens were completely dehydrated with up to 100% ethanol, which was followed by various infiltration times with LR White monomer. Polymerization of the resin was induced by heat, a chemical catalytic procedure (accelerator), or ultraviolet (UV) light. Paraffin, as well as semithin and ultrathin LR White sections were incubated with antibodies reacting to antigens located on the cell surface (stage-specific embryonic antigen-1; SSEA-1), within the plasma membrane (myelin basic protein), in the cytosol (HNK-1, S100 protein), in the cytoskeleton (GFAP, vimentin, neurofilament protein, INT-FIL), and in the extracellular matrix (laminin). All of the examined antigens were immunocytochemically detectable in paraffin-embedded material, while the carbohydrate moieties, HNK-1 and SSEA-1, were not immunoreactive in LR White sections. However, in cryostat sections processed for pre-embedding immunoelectron microscopy, the HNK-1 epitope and SSEA-1 were immunolabelled. Polymerization carried out under UV light led to better structural preservation of brain tissue than resin cured with heat or catalyst. The length of prior infiltration with monomer apparently had no effect on tissue preservation. Consequently, UV light-induced polymerization of LR White gives acceptable morphology of brain tissue. However, the use of this acrylic resin is restricted to the detection of some CNS antigens only.

Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.

Ultrastructural Visualization of Human Papillomavirus DNA in Verrucous and Precancerous Squamous Lesions

Human papillomavirus (HPV) DNA was ultrastructurally localized by the non-isotopic in situ hybridization technique in formalin-fixed, paraffin-embedded specimens of verruca vulgaris of the skin, condyloma acuminatum of the penis and severe dysplasia of the uterine cervix. Biotinylated DNA probe cocktails were employed for the visualization of HPV-DNA, types 6 and 11 (HPV 6/11) and types 16 and 18 (HPV 16/18). The papillomavirus genus-specific antigen was also visualized by pre-embedding immunoelectron microscopy using rabbit antiserum. In verruca vulgaris, HPV antigen-positive 50-60 nm-particles of mature viral size were observed in the nuclei of the granular cells and parakeratotic cells with perinuclear haloes, whereas HPV 6/11 and HPV 16/18 DNA were negative. In condyloma acuminatum, the nuclei were positive for the HPV antigen and HPV 6/11 DNA, but were negative for HPV 16/18 DNA. More cells were labeled for the viral DNA than for the viral antigen. The ultrastructural observation indicated the presence of the naked (plasmid) viral DNA as fine particles sized 15-20 nm. In the dysplastic cervical mucosa, dot-like positivity of HPV 16/18 DNA was recognized. The HPV antigen and HPV 6/11 DNA were undetectable. HPV 16/18 DNA was localized in part of the nuclear chromatin. This pattern of localization may suggest integration of the viral DNA into the host cell DNA.

Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantages of microwave fixation.

Glutathione peroxidase (GSH-PO), a highly soluble, selenium-dependent enzyme metabolizing lipid peroxides, is allegedly distributed in both the cytosol and mitochondria. With the pre-embedding method of immunoelectron microscopy for GSH-PO employing conventional immersion-fixation, the nuclei of rat hepatocytes stain positively, whereas mitochondria are negative. Such observations are inconsistent with the results of biochemical and immunoblot analyses using isolated subcellular fractions. In the present study, we employed the combination of microwave irradiation and fixation in 4% paraformaldehyde (PFA), with or without 0.1% glutaraldehyde (GA), to enhance the accuracy of ultrastructural localization of GSH-PO in rat liver. A small block of liver was irradiated by microwave for 10 sec in cold cacodylate-buffered 4% PFA containing 0.1% GA. After further immersion of the tissue in 4% PFA at 4 degrees C for 1-6 hr, the standard procedure for pre-embedding immunoelectron microscopy was employed. We observed partial inhibition of artifactual diffusion of cytosolic GSH-PO into the nuclei and consistent GSH-PO localization in mitochondria. Dual localization of this enzyme in the cytosol and mitochondria of normal rat hepatocytes was thus confirmed.

Ultrastructural localization of neuropeptide FF, a new neuropeptide in the brain and pituitary of rats

The octapeptide FLFQPQRF-NH 2 or neuropeptide FF (‘F8Famide’; ‘FMRFamide-like peptide’; ‘morphine-modulating peptide’) has been isolated from the bovine brain. In this study, the ultrastructural localization of neuropeptide FF-like immunoreactivity was examined with pre-embedding immuno-electron microscopy in the nucleus of the solitary tract and in the posterior lobe of the pituitary gland of an adult rat. Neuropeptide FF-like immunoreactivity was detected only in neuronal structures of the medial and commissural nuclei of the solitary tract and in the neurohypophysis. In the medulla, the peroxidase-antiperoxidase reaction product was localized in large (100 nm) dense-cored vesicles and in the cytoplasm of the neuronal perikarya, dendrites and axon terminals. In the labeled terminals, small (50 nm) clear vesicles rimmed with the peroxidase-antiperoxidase reaction product were seen. Synaptic contacts of labeled perikarya and dendrites with unlabeled axon terminals were observed. Labeled axon terminals formed contacts with unlabeled dendrites and perikarya. In the posterior lobe of the pituitary gland, neuropeptide FF-like immunoreactivity was localized in nerve terminals frequently associated with blood vessels. The results suggest that neuropeptide FF-like peptides are localized exclusively in neuronal structures and that they are synthesized in cell somata and released from axon terminals. In the brain, neuropeptide FF-like peptides may act as neuromodulators involved in the regulation of autonomic functions. The localization of neuropeptide FF-like immunoreactivity in the neurohypophysis suggests endocrine regulatory functions of these peptides.

Fine structure of substance P-containing nerve fibers in the rat lateral septum; simultaneous localization in pre- and postsynaptic elements

The fine structure of nerve fibers with substance P (SP)-like immunoreactivity in the rat lateral septum (LS) was investigated by preembedding immunoelectron microscopy. SP axon terminals frequently made synapses with non-immunoreactive neuronal soma and dendrites in the LS. Occasionally, two closely apposed nerve endings with SP immunoreactivity were presynaptic to the soma. A small number of immunopositive axon terminals formed synapses not only with neuronal perikarya but also with small dendrites in the vicinity of the perikarya. There were also some SP dendrites in contact with immunoreactive as well as non-immunoreactive nerve endings. These findings may provide a morphological basis for the complexity of SP actions on septal neurons.

Novel localization of a G protein, Gz-alpha, in neurons of brain and retina

Recently, a cDNA coding for a novel G protein alpha-subunit, Gz-alpha, was isolated from a human retinal cDNA library and shown by Northern blot analysis to be expressed at high levels in neural tissues. We have prepared affinity-purified antibodies specifically directed against synthetic Gz-alpha peptides and employed immunohistochemical methods to map the localization of Gz-alpha in human, bovine, and murine retina and brain. By light microscopy, Gz-alpha was localized to the cytoplasm of neurons, with predominant reactivity in ganglion cells of the retina, Purkinje cells of the cerebellum, and most neurons of the hippocampus and cerebral cortex. Reactivity was confined to perikaryon, dendrites, and a very short segment of proximal axons, except for the retinal ganglion cells, in which the axons in the nerve fiber layer showed intense Gz-alpha immunoreactivity proximal to the lamina cribrosa. Pre-embedding immunoelectron microscopy demonstrated the presence of focal Gz-alpha immunoreactivity on the nuclear membranes, endoplasmic reticulum, and plasma membranes of Purkinje cell perikarya and in association with microtubules in their proximal dendrites. Subcellular fractionation studies confirmed the association of Gz-alpha with plasma and intracellular membranes. The localization of Gz-alpha and its unique amino acid sequence suggest that it may have a specialized function in neural tissues.

Microwave-stimulated fixation for histocytochemistry: Application to surgical pathology and preembedding immunoelectron microscopy.

Microwave (MW)-stimulated fixation was applied to surgical pathology and preembedding immunoelectron microscopy. Fresh specimens were microwavedin the fixatives for 15-30sec using a conventional MW oven (500W) containing 200ml of water load. The temperature of fixatives was raised to 35-45°C. After MW radiation, the specimens were kept in the same fixatives for 1-4hr at 4°C or at room temperature as the postfixation procedure, which was important for the completion of chemical fixation of whole specimens. For light microscopical examination, buffered 10% formalin was used. Hematoxylin-eosin staining from paraffin-embedded specimens was completed within 24hr with excellent preservation of morphology and immunoreactivity. For conventional electron microscopy, MW fixation in 2.5% glutaraldehyde-2% paraformaldehyde showed excellent preservation of cell organelles. For immunoelectron microscopy by the preembedding method, 4% paraformaldehyde solution containing 0.1% glutaraldehyde was used, followed by one hr postfixation. Simultaneous improvement of both ultrastructure and antigenicity were obtained without any significant changes of the results. Our results indicate that MW-stimulated fixation with postfixation procedure is a useful tool for various fields of morphological investigations, and its beneficial effects were most remarkable for the preembedding immunoelectron microscopy technique.

Identification of La ribonucleoproteins as a component of interchromatin granules

Monoclonal antibodies raised against the La antigen were used to localize, by preembedding immunoelectron microscopy, snRNPs containing this protein. The results demonstrate that La RNPs are localized in clusters of interchromatin granules, both in Triton X-100-extracted and DNase-digested nuclei. DNase-digested salt-extracted nuclei contained, in addition, labeled structures identified as perichromatin granules and fibers. A close association of labeled granules with the nucleoli was also observed. Digestion of nuclei with DNase yielded residual scaffolds of intermediate filaments and nuclear lamina devoid of interchromatin granules and La immunostaining. Release of the La antigen was tested in the presence of ATP and cytochalasin B. Only cytochalasin was effective, suggesting a role for nuclear actin in anchorage of snRNPs.

A method permitting precise trimming of resin-embedded tissue for ultrathin sectioning in pre-embedding immunoelectronmicroscopy

Due to the high complexity of the mammalian central nervous system, sampling of immunohistochemically processed brain tissues for electronmicroscopy requires an accurate and reliable technique. For this reason, the flat-embedding method, which allows light microscopical examination of tissue before sampling, is generally employed. Because of the osmification process, however, the tissue is blackish and opaque which hampers light microscopical selection of tissue areas of interest. We have found that tissue translucency is highly improved by an osmification process using an osmium tetroxide-ferrocyanide mixture. We describe a transilluminated chuck that enables visualization of immunostaining in specimens mounted on a trimming instrument, thus allowing for extremely precise sampling of the tissue.

Immunoelectron microscopic study of distribution of T cell subsets in oral lichen planus

Monoclonal anti-CD4, anti-CD8, and anti-CD18 antibodies were applied in avidin-biotin-peroxidase complex staining using a pre-embedding immunoelectron microscopy technique. Although most of the local T cells in situ were of CD4+ subtype, local CD8+ cells generally had a lower nucleus/cytoplasm ratio and contained more cell organelles than CD4+ cells. This suggests a local activation of CD8+ subpopulation, rather than activation of the numerically predominant CD4+ cells. Topographical analysis disclosed that all lymphocytes, regardless of location, were CD18+ and that most of the CD8+ cells were located subbasally and intraepithelially, whereas CD4+ cells often occurred in small clusters deeper down in the subepithelial lymphocyte-rich band. Furthermore, CD8+ cells were often in close contact with macrophages, whereas CD4+ cells were in some instances in direct contact with plasma cells. This indicates that CD4+ cells may be involved in T cell-dependent B cell-mediated immunoglobulin synthesis, whereas CD8+ cytotoxic lymphocytes and tissue macrophages may be involved in the local pathogenetic process leading to basement membrane alterations.

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and pre-embedding immunogold labelling.

A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.

Isolation and Characterization of Paraflagellar Proteins from Trypanosoma cruzi

Two different Trypanosoma cruzi polypeptides, with masses of 70 and 68 kDa were purified and characterized in this work. These two polypeptides designated PAR 1 and PAR 2, respectively, co-purified during each step of the isolation procedure and were found to be located exclusively in T. cruzi flagella by indirect immunofluorescence. A pre-embedding immunoelectron microscopy procedure, with a gold-tagged secondary antibody, permitted direct identification of PAR 2 as a component of the T. cruzi paraflagellar rod. PAR 1 and PAR 2 were found to be immunologically distinct and showed no cross-reactivity with actin, tubulin, intermediate filament proteins, or other proteins present in mammalian cells. The results presented indicate that PAR 1 and PAR 2 are the major components of T. cruzi paraflagellar filaments, and that these filaments have no counterpart in mammalian cells.

Reassessment of enkephalin (ENK)-containing afferents to the rat lateral septum with reference to the fine structures of septal ENK fibers

Using a combination of horseradish peroxidase (HRP) histochemistry and immuno-β-galactosidase staining, and pre-embedding immuno-electron microscopy, the present study was intended to re-examine the origins of enkephalin-like immunoreactivity (ENKI) in the rat lateral septum (LS), and to show the fine structures of septal ENKI fibers. Following HRP injection into the LS, double-labeled cells which contained a homogeneous blue reaction product of ENKI and a black or brown granular reaction product of retrogradely transported HRP were identified in 4 discrete brain regions: perifornical hypothalamic area at the level of the paraventricular nucleus (PeF); posterior part of the anterior hypothalamic nucleus (AHP); bed nucleus of the stria terminalis, medial division, posterolateral part (BSTMPL); and dorsal hypothalamic area (DA). Immuno-electron micrographs demonstrated that some of the ENKI terminals in the LS form synapses with the soma and dendrites of septal neurons devoid of ENKI, though ENKI dendrites postsynaptic to non-immunoreactive terminals were also seen in the LS. These findings suggest that a large proportion of septal ENKI fibers have their origins in the above regions (PeF, AHP, BSTMPL, DA) different from the previously considered one, and they further provide a morphological basis for the postsynaptic inhibitory effects of enkephalins on septal neurons.