Postembedding immunogold electron microscopy for S100, tubulin, and cytoskeletal proteins in an amelanotic malignant melanoma

A postembedding Protein A-colloidal gold technique was used for the ultrastructural immunocytochemical investigation of S100, tubulin, and cytoskeletal proteins in a Lowicryl K4M-embedded melanoma with numerous microtubules. S100 protein was localized in the cytoplasm and the nuclei of tumor cells. Melanosomes were not labeled with S100. Perinuclear intermediate filaments and filaments in cytoplasmic processes reacted positively for vimentin. The straight, rod-shaped, parallel intracisternal microtubules failed to react with antisera to tubulin, S100, vimentin, cytokeratin, neurofilaments, desmin, glial fibrillary acidic protein, and immunoglobulin light chains. These results give an improved correlation between the ultrastructural and immunocytochemical characteristics of the tumor, confirm the melanocytic origin of it, and demonstrate the application and usefulness of the postembedding immunogold method in the investigation of the protein composition of subcellular structures.     

Radial glial cells derived from the neonatal rat spinal cord: morphological and immunocytochemical characterization

Radial glial cells are transiently bipolar cells in the developing central nervous system, best known for their role in guiding migrating neurons. The aim of the present study was to investigate phenotypic characteristics of these bipolar precursor cells in a mixed glial cell culture system derived from the rat neonatal spinal cord. Morphological characterization was assessed by cell-specific immunocytochemical markers (nestin, vimentin, 3CB2) and transmission electron microscopy. Our study yielded substantial evidence showing that the bipolar cells exhibit immunocytochemical and ultrastructural features of radial glial cells. Immunohistochemistry of the neonatal rat spinal cord using the same cell-specific markers suggested these cells are likely derived from the subependymal zone, ventral commissure, and dorsomedial septum. We believe our data recommend this mixed glial culture system to be a valuable tool in studying radial glial cells in vitro.     

Immunocytochemistry of neuronal and glial markers in retinoblastoma

Summary An immunocytochemical study of 30 retinoblastomas was carried out using antibodies to neuronal and glial markers. The tumours were found to react with antibodies to neuron-specific enolase (NSE), a marker for neuronal elements, and S-100 and glial fibrillary acidic protein (GFAP), both of which are proteins present in glia. Two distinct cell populations were found within the tumour: the first, composed of anaplastic tumour cells at various stages of differentiation, showed both NSE and S-100 immunoreactivity; the second cell type, which immunostained for S-100 and GFAP, resembled mature glial cells. The results of this study indicate that the retinoblastoma may arise from a pluripotential primitive cell partially retaining neuronal and glial characteristics.     

Heterogeneity in spinal radial glia demonstrated by intermediate filament expression and HRP labelling

Summary Considerable evidence indicates that radial glial cells play an active role in guiding growing neurites during development of the vertebrate CNS. In this paper we describe subpopulations of radial glia in the spinal cord of the axolotl. Amphibians maintain radial glia throughout life, and subpopulations are described using anatomical criteria following filling of individual cells with horseradish peroxidase and immunocytochemical staining with a range of intermediate filament antibodies.Radial glial cells in specific regions of the spinal cord stain with a range of antibodies specific to human keratins 8 and 18, and to glial fibrillary acid protein (GFAP). Some of these antibodies show selective staining localized to specific regions of individual glial cell processes. Immunoblotting analysis indicates that two keratins are present in the axolotl CNS corresponding to the two earliest embryonic keratins of vertebrates, keratins 8 and 18. Comparisons of molecular weight indicate that these may correspond to keratins identified inXenopus laevis, the genes of which have been cloned. Axolotl GFAP is also identified in Western blots and may be present in two forms of differing molecular weight.These results are discussed in terms of the likely role of radial glial cells, and comparisons are drawn between the keratin and GFAP types seen, in the axolotl spinal cord and of those in other vertebrate groups.     

Characterization of normal human brain cultures. Evidence for the outgrowth of leptomeningeal cells

To establish the histogenetic identity of the predominant cell type in monolayer cultures of normal human adult brain, eight brain specimens were placed into culture and characterized according to cell kinetics, karyotype, antigenic expression, and ultrastructural features. The protein profiles of both the cell layer and the medium were analyzed in selected cultures using sodium dodecyl sulfate polyacrylamide gel electrophoresis and diethylaminoethyl cellulose chromatography. All cultures displayed a limited life span in vitro; marked contact inhibition at confluence; a normal karyotype; an intracytoplasmic and extracellular glycoprotein profile consisting of fibronectin, procollagen type III, laminin, and collagen type IV; specialized intercellular junctions; and interstitial collagen chain synthesis. All of these features were identified in our previous study of human leptomeningeal cultures. The results of immunocytochemical staining for glial fibrillary acidic protein were negative in all cultures of normal human brain, except in early passages in two cultures, which lost the glial cell marker during subsequent passages; immunostains for vimentin were positive in all cells in all cultures. These results support the hypothesis that, in this study, cultures derived from normal human brain are not of glial origin. Our findings also suggest that glial cells are less well-suited to monolayer growth under our culture conditions than are other cell types in enzyme-dissociated brain tissue placed in culture, especially leptomeningeal cells. The identification of leptomeningeal cells as the predominant cell type in normal human brain cultures may prove useful in attempts to foster the growth of human glial cells by culturing brain samples under conditions that prohibit the growth of leptomeningeal cells. Under such conditions, astrocytes, oligodendroglia, and ependymal cells could be isolated with greater ease and cultured separately. These purified cultures of different glial cell types would then provide a more relevant in vitro model for studying human neurological diseases.     

Gliogenesis in organotypic tissue culture of the spinal cord of the embryonic mouse. I. Immunocytochemical and ultrastructural studies

Summary The technique of organotypic tissue culture offers an opportunity to observein vitro complex interactions among glial cells and neurons, leading to the formation of myelin. In the present and accompanying work a combined ultrastructural, immunocytochemical and autoradiographic approach was used in a detailed study of the process of gliogenesis. Using immunocytochemical and ultrastructural criteria, differentiation along the oligodendroglia cell line is seen to be initiated a few days later than along the astroglial line. The sequence and timing of oligodendroglial differentiation both ultrastructurally and chemically follow those describedin vivo. Formation of myelin has been demonstrated only by oligodendrocytes in which there is continuity between the perikaryal plasmalemma and myelin membranes. Oligodendroglial maturation culminated with the formation of light, medium and dark oligodendrocytes. The periodic acid Schiff-positive, glial fibrillary acidic protein (GFAP)-negative processes of radial glial cells at explantation become GFAP-positive within 3 days, as describedin vivo. Many of the astrocytes appear to have been derived from radial glial cells. Large numbers of dark glial cells, similar to the so-called intermediate glial cells, were seen. These were found to be astrocytes whose appearance probably reflected reaction to explantation-induced injury.     

Astrocytes and microglia in human brain share an epitope recognized by a B-lymphocyte-specific monoclonal antibody (LN-1)

A B-lymphocyte-specific mouse monoclonal antibody, LN-1, recognizes two morphologic classes of glial cells in human brain. The nature and duration of tissue fixation and processing are critical in the detection of the two cell types. In tissue that is lightly fixed, LN-1 recognizes astrocytes. The astrocytic nature of the LN-1 reactive glial cell was confirmed by cytologic features, tissue distribution, immunoelectron microscopy, double labeling immunofluorescent microscopy, and staining of serial sections with antibodies to glial fibrillary acidic protein. In tissue that is fixed for longer periods or in Bouin's fixative, two glial cell types are recognized: astrocytes and microglia. The identity of the latter cell type as microglia was confirmed by morphologic features, tissue distribution, immunoelectron microscopy, and double staining with monoclonal antibodies or lectins to macrophage markers, including class II major histocompatibility antigens. The two cell types had different disposition in senile plaques of elderly individuals and of those with Alzheimer's disease. Astrocytes were present at the periphery of the plaques, whereas microglial cells were centrally placed, often in juxtaposition to amyloid. The results are discussed with respect to ontogeny of glial cells and the ability of monoclonal antibodies to recognize epitopes on unrelated proteins.     

Immunocytochemical localization of NOVH protein and ultrastructural characteristics of NCI-H295R cells

In this study we present biochemical and immunocytochemical results on NOVH protein secretion and localization in NCI-H295R cells, as well as results on the ultrastructural characteristics of NCI-H295R cells. NCI-H295R cells were characterized by small quantities of rough and smooth endoplasmic reticulum, many free ribosomes, large nuclei with prominent nucleoli, numerous elongated mitochondria, a few Golgi complexes, and a small number of lipid droplets. Large numbers of coated pits and coated vesicles were present, but no secretory granules or exocytotic profiles were seen. Best ultrastructural preservation of NCI-H295R cells was achieved when fixation was done directly on the culture dishes and the cells were detached by scraping. Our biochemical results showed that NCI-H295R cells secreted large amounts of NOVH protein. The immunocytochemical localization of NOVH protein showed that the protein was localized in the cytoplasm, the plasma membrane and the nuclear envelope. This localization pattern, along with the ultrastructural and biochemical findings raise interesting questions on the function(s) and the mode of secretion of NOVH protein.     

Ultrastructural features of medullary chromaffin cell cultures

The ultrastructural organization on the fourth day of culture of chromaffin cells isolated from the bovine adrenal medulla was characterized based on electron microscopic and morphological analysis. We established that medullary chromaffin cells could be divided into four morphologically different subtypes. Most cells (49.1% of those examined) had a dense cytoplasm and fine dense granules. Cells with dense cytoplasm and large granules represented a second type of chromaffin cell (21.1%). Cells of the third type had a light cytoplasm, granules with a light halo and a well-developed Golgi apparatus (26.3%). The fourth type of chromaffin cell was characterized by moderately dense cytoplasm with well-expressed varicose rough endoplasmic reticulum (about 3.5%). Among concomitant cell types, cortical adrenal cells from the zona fasciculata and zona glomerulosa, epithelial cells, fibroblasts, lymphocytes, brown lipoblasts and glial Schwann cells were present. Morphological analysis implies that cells with dense cytoplasm and fine granules and those with light cytoplasm and haloed granules (75.4% in total) are adrenaline-containing cells, whereas the cells with dense cytoplasm and large granules (26.3%) contain noradrenaline. Cells with moderately dense cytoplasm and varicose reticulum share common morphological properties with classical glandular cells and, by their properties, were closer to noradrenaline-containing cells.It is concluded that chromaffin cells, which are the main cell type among cultured cells from adult bovine adrenal medulla, are morphologically quite heterogeneous. Other cell types of different nature may also be present in the culture and can locally influence the properties of the investigated medullary chromaffin cells used in electrophysiological experiments.     

The fine structure of gliomatosis cerebri

Summary The ultrastructural features of five biopsies of gliomatosis cerebri (GC) are described. Four main types of tumour cells are seen: anaplastic astrocytes poor in organelles with a variable amount of glial microfilaments; atypical oligodendrocytes with scanty cytoplasm in which microtubules are present; intermediate forms with aboundant cytoplasm rich in organelles, with microtubules and microfilaments; and small cells with round nuclei and a very scanty rim of cytoplasm. In two cases several concentrically folded cytoplasmic lamellae of glial processes were arranged either around themselves or around the perikaryon of other cells. This ultrasructural study indicates that GC is a neoplastic process of small undifferentiated elements, transitional forms of astroglia (to oligodendroglia) and anaplastic cells of astrocytic origin in all stages of development.Dedicated to Prof. Dr. Lennert on occasion of his 65th birthdayOn leave from the IIBM-UNAM Mexico with a DAAD fellowship     

The cytoskeleton in tumor cells

During the past few years several laboratories investigated the occurrence of cytoskeletal components in epithelial and mesenchymal cells by electron microscopy and/or immunocytochemical methods in a number of tumor types growing in vitro or in the body. Since it is well established that antibodies to different intermediate-sized filament proteins can distinguish cells and tissues of epithelial, mesenchymal, muscle, astrocytic and neural origin special attention has been paid to the behaviour of these filaments in neoplastic cells recently. While the organisation of the cytoskeleton in tumor cells growing in vitro is very variable, regularities relevant for the diagnosis and the determination of the histogenetic origin of tumors have been observed in tumor cells growing in the body. In general, ultrastructural and immunological features of intermediate filaments are maintained during neoplastic transformation in the body. Thus immunofluorescence microscopy with antibodies to cytoskeletal proteins is a powerful tool for the classification and differential diagnosis of tumors, especially for the distinction between epithelial and mesenchymal tumors, including metastases. The concept that presence of an excess of contractile proteins such as actin is an important prerequisite for the metastatic spread of malignant cells has not been unequivocally supported by more recent results. However, an accumulation of various types of intermediate filaments (e.g. prekeratin, vimentin, acidic glial fibrillar protein) has been shown in different tumor types. The further elucidation of this alteration could contribute to a better understanding of the molecular mechanisms of neoplastic cell transformation.     

In vivo CNS remyelination: HNK-1 labels newly differentiated oligodendrocytes but not precursors

Summary The possibility that HNK-1 antibody might identify oligodendrocyte precursorsin vivo was investigated using a combined immunocytochemical and ultrastructural analysis of remyelination in the cat optic nerve after demyelination induced by local injection of antiserum against galactocerebroside. HNK-1 antibody, in addition to antisera to galactocerebroside, glial fibrillary acidic protein, vimentin and myelin basic protein, was applied to early and late premyelinative lesions within which we have recently identified a putative precursor for the remyelinating oligodendrocyte termed the small glial cell. HNK-1 labelled oligodendrocytes in normal optic nerve and newly differentiated oligodendrocytes within lesions but did not label the small glial cells.     

Elektronenmikroskopische Untersuchungen zur Zelldifferenzierung in Prostata-Carcinomen

Summary 20 confirmed human prostatic carcinomas of various histological types were studied by electron microscopy. As a result of the ultrastructural findings it was possible to define characteristic cell types which are normally present in prostatic carcinomas irrespective of their histological differentiation. As these cell types are frequently situated side by side, the prostatic carcinoma acquires a characteristic variegated appearance. Three main cell types can be distinguished. 1. A cell with a pale, euchromatic, round nucleus, very prominent nucleolus, and a narrow layer of bright cytoplasm with few organelles. This cell shows no secretory activity and has to be classified as a poorly differentiated or embryonic cell. It may be assumed that these cells have a high proliferative activity. 2. A cell with dark, pleomorphic, heterochromatic nucleus and a broad layer of dark cytoplasm containing numerous organelles. This cell type is apparently functionally stimulated and active. The disproportionate increase and arrangement of the organelles, however, makes it morphologically atypical: These alterations seem to result from an irregular process of enzyme synthesis and secretion. 3. A vacuolated cell: this cell has a dark, pyknotic nucleus and a broad layer of subtotally vacuolated cytoplasm. Probably it refers to a nonfunctional, hypersecretory and degenerative tumour cell. Transitional cell forms are not unusual. Sometimes carcinoma cells are visible in tumourous glands, and are so highly developed and well differentiated that the ultrastructural findings by themselves give no answer in the question of malignancy.Basal cells were seen in five of the carcinomas investigated, mainly in tumours with cribriform or solid patterns. The cytoplasm of these cells shows few organelles. Bundles of cytoplasmatic filaments are often present. It is uncertain whether these cells are of myoepithelial type.The knowledge of the described cell variants will be of clinicopathological importance if therapeutic effects on prostatic carcinoma are estimated cytomorphologically at the ultrastructural level.     

Antibody microarray for correlating cell phenotype with surface marker

To correlate cell surface markers with the cell phenotype, an antibody microarray prepared by covalently immobilizing antibodies onto a cellulose membrane and subsequent immunocytochemical staining were employed. The direct binding assay of a lymphoblastic leukemia cell line on the microarray showed that the immobilized antibody served to capture cells expressing the specific antigen. The density of bound cells increased linearly with an increasing content of antigen-expressing cells in suspension. The method was further applied to the analysis of surface antigens expressed on neural stem cells. A binding assay was performed with neural cells obtained from the neurosphere culture of the rat fetal striatum on a microarray spotted with eight kinds of antibodies and four different proteins, followed by immunocytochemical staining of cells bound to the microarray using antibodies to the intracellular markers of immature (nestin and vimentin) and mature (beta-tubulin III and glial fibrillary acidic protein) neural cells. As a result, the phenotype of bound cells could be correlated to surface antigen expression, which illustrated the potential of the solid-phase cytometry developed here for the identification of surface markers.     

Clear cell chondrosarcoma: case report and ultrastructural study

A case of clear cell chondrosarcoma in a Chinese patient is described. The clear cells showed strongly positive S-100 protein immunoreactivity. Ultrastructurally 2 types of chondroid cells were demonstrated. One type appeared more primitive with abundant electron-lucent cytoplasm and sparse organelles. The other type of cell was more differentiated with presence of microvilli and numerous dilated cisternae of endoplasmic reticulum. Previous ultrastructural studies on these lesions were reviewed and compared with the present findings.     

Ultrastructural Features of Neuroendocrine Differentiated Carcinomas of the Breast

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.     

Distribution of glial fibrillary acidic protein and glutamine synthetase in human cerebral cortical astrocytes — a light and electron microscopic study

Summary Human cerebral cortex was studied immunocytochemically by light and electron microscopy using antibodies against glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). Glial fibrillary acidic protein-positive cells and processes were present in both cortex and white matter, but in contrast glutamine synthetase-positive cells and processes were present only in cortex. Cell bodies which contained glutamine synthetase had typical ultrastructural features of protoplasmic astrocytes. Glutamine synthetase-positive processes were often present near asymmetrical synapses in the neuropil. These processes often contained mitochondria, but not glial filaments, and were different from unlabelled astrocytic processes, which seldom contained mitochondria, but had large numbers of glial filaments. Glutamine synthetase immunoreactivity therefore affords a means of distinguishing between these two types of astrocytic processes in the human cerebral cortex.     

人胚胎海马发育的形态学研究──Ⅱ．神经细胞与神经胶质细胞的分化

利用Ｈ－Ｅ和Ｎｉｓｓｌ染色法、免疫细胞化学法、Ｇｏｌｇｉ镀银法及透射电镜技术，对６０例６周至足月人胚胎海马神经细胞及神经胶质细胞的分化、发育进行了观察。结果表明；神经细胞及神经胶质细胞均由未分化细胞转化而来。未分化细胞、神经细胞和神经胶质细胞在对硝酸银的嗜染性、免疫细胞化学特征及超微结构特征等方面都有显著差别。神经细胞为神经元特异性烯醇化酶（ＮＳＥ）阳性细胞，主要有锥体细胞、颗粒细胞和篮状细胞等。星形胶质细胞和放射状胶质细胞为胶质原纤维酸性蛋白（ＧＦＡＰ）阳性细胞。未分化细胞体积小、球形、胞质少，为ＮＳＥ及ＧＦＡＰ阴性，硝酸银镀染也不着色；透射电镜下未分化细胞核异染色质丰富，胞质内缺乏特化的细胞器，但糖原含量丰富。胚胎早期室管膜神经上皮细胞及由此迁徙而来的中间层细胞均由未分化细胞构成。星状胶质细胞和放射状胶质细胞出现较早，第１１周开始出现于室管膜下的室床及海马伞部；随胎龄增加，单位面积垦状胶质细胞数量增加，１７周后维持在相对稳定状态，并且均匀分布于海马各个部位与区域。１５周后中间层细胞陆续开始分化为锥体细胞和颗粒细胞，到足月胚胎锥体层及颗粒层不再有未分化细胞。     

Expression of the p53 protein in a spectrum of astrocytic tumours.

Many human cancers are characterized by mutations of p53, a nuclear phosphoprotein which controls elements of the cell cycle. Turnover of p53 in normal cells is rapid, and the minute quantities of protein that are usually present are not detected by immunocytochemical methods. Mutations of the p53 gene in tumour cells are associated with a slower turnover and subsequent accumulation of the protein in both nucleus and cytoplasm. Genetic abnormalities of the short arm of chromosome 17, which is the site of the p53 gene locus, are a feature of astrocytic tumours. Using a panel of five antibodies to p53 and a standard immunocytochemical method, we found detectable quantities of p53 in the cells of 3/16 diffuse astrocytomas, 8/14 anaplastic astrocytomas, and 24/34 glioblastoma multiforme. Progression of one patient's tumour from a diffuse to an anaplastic astrocytoma was characterized by the accumulation of p53. The more malignant histological features of anaplastic astrocytoma and glioblastoma multiforme appear to be reflected by a greater incidence of p53 accumulation.