Ultrastructural identification of protein bodies, cellular markers of human catecholamine neurons, in a temporal lobe ganglioglioma

A temporal lobe ganglioglioma, surgically removed from an 8-year-old body, and a human brainstem at the level of locus coeruleus (LC) were processed for light microscopy (LM), with formalin fixation and paraffin embedding, and for electron microscopy (EM) with glutaraldehyde fixation, potassium permanganate postfixation, phosphotungstic acid-hematoxylin block-staining, and epoxy-resin embedding. The paraffin sections were stained with toluidine blue O/rhodamine B and observed under epi-fluorescence. The thin sections for EM were viewed directly without further staining. The neuronal neoplastic cells of ganglioglioma and the neurons of LC are known to produce catecholamines. Both also contain spherical protein bodies (pb), cellular markers that identify catecholamine neurons in humans. The ultrastructural characteristics of the pb in LC were compared with those of the pb in neoplastic ganglion cells. These bodies had an identical ultrastructure, in both tissues, consisting of electron-lucent core surrounded by an electron-dense thin rim. The rhodamine B-stained sections also emphasized the identical morphology of the pb in ganglioglioma and LC. Based on the EM comparison, these brightly fluorescing spherical bodies are ideal markers for identifying in LM, the clusters of large neoplastic cells, representing neurons, which are the most important clue to the correct diagnosis of gangliogliomas.     

Langerhans cells in nasal mucosa of patients with grass pollen allergy.

Langerhans cells (LC) are known to be present in squamous epithelia of the human body. They are dendritic cells (DC) and characterized by the presence of Birbeck granules (BG). In previous studies, DC positive for CD1a and HLA-DR were found in the cylindrical epithelium and the lamina propria of the nasal mucosa. In our study, more CD1a cells occurred in the allergic patients than in the non-allergic controls. In a combined light microscopy (LM) and electron microscopy (EM) study, biopsies of nasal mucosa in allergic patients were studied. We used monoclonal antibodies against CD1a and HLA-DR, to identify DC in LM cryostat sections. The presence of BG identified most of the intra-epithelial DC as LC on the EM level, whereas a minority of DC in the lamina propria also contained BG. The ultrastructure of LC and DC in the ciliated cylindrical epithelium and the lamina propria is compared.     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

乙醇和丙酮对绿色荧光蛋白强度作用的对比

Thegreenfluorescentprotein(GFP)hasbeen widelyacceptedasahighlyusefultoolinfluorescence studiesoflivingcells.Itwasfirstfoundincellcyto plasmofjellyfish[1,2]andisanextremelystableprotein of238aminoacids.Itwasreportedthatthefluorescent propertiesoftheproteinwereunaffectedbyprolonged treatmentwith6MguanidineHCL,8Mureaor1% SDS,andtwodaystreatmentwithvariousproteases suchastrypsin,chymotrypsin,papain,subtilisin, thermolysinandpancreatinatconcentrationsupto1 mg/mlfailtoaltertheintensityofGFPflu…     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

Immunohistochemical analysis of human lymphomas with monoclonal antibodies to B cell and Ia antigens reactive in paraffin sections

Monoclonal antibodies (MoAbs) to B cell- and T cell-specific antigens uniformly have been restricted in their use to cell suspension or frozen section techniques because the antigens that they identify are either masked or lost in the fixation or paraffin-embedding processes. Antiimmunoglobulin antisera, although readily identifying cytoplasmic immunoglobulin in paraffin sections, has not been as useful as originally hoped since the majority of B cell lymphomas express surface immunoglobulins, which requires cell suspensions or frozen sections for detection. Because cell suspension procedures disrupt tissue architecture and frozen section techniques grossly distort morphology, neither method allows the combination of optimal morphologic and immunologic classification of lymphomas. We recently reported two MoAbs, LN-1 and LN-2, that react with B cells in paraffin sections. LN-1 reacts with the surface membrane and cytoplasm of germinal center B cells. LN-2 reacts uniquely with the nuclear membrane and cytoplasm of mantle zone and germinal center B cells and interdigitating histiocytes. We have also identified a new MoAb, LN-3, that reacts with the HLA-DR antigen in paraffin sections. We now report the use of LN-1, LN-2, and LN-3 in the analysis of paraffin sections from 58 non-Hodgkin's lymphomas and 15 cases of Hodgkin's disease. The types of cells reactive with these MoAbs in neoplastic lymphoid proliferations largely recapitulate their benign morphologic and immunologic counterparts. As a panel, LN-1, LN-2, and LN-3 were reactive with 98% of B cell lymphomas, and LN-1 and LN-2 were negative on all T cell lymphomas. In addition to identifying the cell of origin of these malignant proliferations, these MoAbs were also useful for identifying architectural features in neoplastic lymph nodes. Thus, these reagents provide the ability to assess the immunologic phenotype of neoplastic lymphocytes in conjunction with the critical morphologic criteria requiring paraffin embedding.     

The role of immunohistochemistry, electron microscopy, and ultrastructural cytochemistry in the diagnosis of mixed carcinoma-neuroendocrine neoplasms

We studied four mixed carcinoma-neuroendocrine neoplasms from gastrointestinal tract and pancreas by routine light microscopy (LM), immunohistochemistry (IH), electron microscopy (EM), and ultrastructural cytochemistry (UC). By LM, the individual tumors showed fairly pure neuroendocrine (carcinoid) or epithelial (papillary) patterns, mixed neuroendocrine-carcinoma features and poorly-differentiated tumor in sheets and nests which did not lend itself to morphologic characterization. IH demonstrated mixed expression, within different areas of the same neoplasm, of epithelial antigens (keratins and carcinoembryonic antigen [CEA]) and neuroendocrine markers (neuron-specific enolase [NSE], bombesin and neurohormonal peptides). By EM, each tumor showed ultrastructural features of epithelial and neuroendocrine differentiation which varied substantially in terms of number of cells involved and their distribution; two of the neoplasms showed biphasic differentiation within single cells. The nature of the neurosecretory granules was verified with the uranaffin reaction (UR). This study illustrates the value of combining LM, IH, EM and UC for the identification of mixed carcinoma-neuroendocrine lesions.     

Application of Grimelius argyrophil staining to the study of tumour ultrastructure. II. Effects of resin embedding: Epon

2 methods have so far been proposed for staining samples by the argyrophil technique of Grimelius for electron microscopy. Vassallo et al. (1971) used en bloc staining after fixation with a mixture of glutaraldehyde and formaldehyde, then H?kanson et al. (1971) stained sections from samples previously fixed by double formaldehyde/OsO4 treatment and embedding in resin. Another investigation that concerned glutaraldehyde fixation showed the effect of this fixative in sample staining (Soranzo and Roland 1987). The present experiments demonstrated that Epon intervenes directly in argyrophil staining of sections. With this resin, the staining process lasted longer than with paraffin embedding materials and offered no guarantee of positivity. Granules with a double structure and dense core displayed different locality of argyrophilia before and after embedding.     

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues.

Routine fixation and paraffin embedding destroys many hematopoietic and lymphoid differentiation antigens detected by flow cytometry or frozen section immunohistochemistry. On the other hand, morphologic evaluation is difficult in flow cytometric or frozen section studies. A simplified three-step plastic embedding system using acetone-fixed tissues embedded in glycol-methacrylate (GMA) resin has been found to provide both excellent morphologic and antigenic preservation. With our system, a wide variety of antigens are detected in plastic sections without trypsinization or prolonged embedding procedures; pan-B (CD19, CD22), pan-T (CD7, CD5, CD3, CD2), T-subset (CD4, CD8, CD1, CD25) markers as well as surface immunoglobulin and markers for myeloid and mononuclear-phagocyte cells are preserved. In summary, modifications of plastic embedding techniques used in this study simplify the procedure, apparently achieve excellent antigenic preservation, and facilitate evaluation of morphologic details in relation to immunocytochemical markers.     

Lysosomal accumulation of SCMAS (subunit c of mitochondrial ATP synthase) in neurons of the mouse model of mucopolysaccharidosis III B

The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme alpha-N-acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6-months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of "zebra bodies", well known but little understood inclusions in the brain of patients with mucopolysaccharidoses.     

The demonstration of cell surface antigens on T cells, B cells and accessory cells in paraffin-embedded human tissues

This paper describes a method for processing fresh tissue that allows immunohistological analysis on paraffin sections. The method is based on the use of periodate-lysine-paraformaldehyde fixation. The effects of variation in fixation time, concentration of paraformaldehyde, dehydration, clearing, wax embedding and enzyme treatment of cut sections were examined. An optimal processing procedure was established that retains good tissue morphology and allowed 21 out of 27 monoclonal antibodies tested to be used successfully on paraffin sections to identify all major cell subpopulations by their membrane antigenic characteristics. The value of this approach in studying the immunopathology of potentially dangerous infectious diseases and in leukaemia/lymphoma diagnosis is discussed.     

Using Kryofix as alternative for formalin results in more optimal and standardized immunostaining of paraffin sections.

Although used for over one century formalin has several disadvantages which Kryofix, an alternative fixative for paraffin blocks used in Leiden for 6 years, does not have. In this study the effects of Kryofix on tissue regarding immunohistochemistry are compared with those of buffered formalin. All markers studied showed enhanced staining in the Kryofix blocks after 4 hours of fixation, whilst in some cases the immunostaining of the formalin blocks was even negative. For all markers, the sera could be further diluted for the Kryofix sections, for some with as much as a factor 20, enhancing the cost-effectiveness of the method. We established that for formalin, the fixation time strongly influenced the results. For Kryofix there was no time factor: the immunostaining results of 1 hour fixation were identical to those after 3 months of fixation. This study shows that by this method of fixation, in which there is no cross-linking of proteins, immunostaining of paraffin sections is optimized and standardized.     

Light chain deposition disease of the kidney. Morphological aspects in 24 patients

Renal biopsies and autopsy specimens of 23 patients with light chain deposition disease (LCDD) and one with only heavy chain deposits, were studied by light (LM) and electron microscopy (EM) as well as immunohistology (IH). Thirteen patients had multiple myeloma; 1 had lymphoma, and 1 chronic myeloid leukaemia with polycythaemia vera. In nine patients, no lymphoproliferative disease was identified. The LM lesions most suggestive of LCDD, nodular glomerulosclerosis (NS) and thickening and wrinkling of the tubular basement membranes (TBM), were present in only ten and 13 patients, respectively. In five of seven specimens without NS or TBM thickening by LM, EM was negative, indicating a limited value of EM in confirming the diagnosis. Renal amyloidosis was not identified, but in one patient amyloid in the heart and tongue was seen at autopsy. One patient had both granular and extensive glomerular non-amyloid fibrillary deposits. In two patients myeloma casts were identified. Twenty-one patients showed renal LC immune reactivity, 1 had both alpha heavy and lambda LC, 1 had only detectable gamma heavy chain. One biopsy was negative by IH, but had characteristic electron dense deposits. In six patients with immune reactivity to LC, no electron dense deposits could be identified by EM. This study emphasizes the spectrum of renal changes by LM and EM in LCDD, the frequent lack of consistency between deposits detected by IH and EM and the difficulty in coming to a definite diagnosis without LM, EM and IH. The results of this study and examination of the literature indicates that extensive morphological changes are more often present in kappa than in lambda LCDD.     

Microwave fixation: in situ tick (Acari: Ixodidae) histoanatomy, thin sectioning of tick tissues, and antigen preservation in mouse spleen

Microwave irradiation was used for the fixation of eggs, nymphs, and adult Boophilus spp. ticks. Although optimal temperatures for fixation of the different tick stages varied, heating to 58 degrees C of adult ticks submerged in either PBS or fixative was found to be sufficient. After microwave fixation, whole adult ticks, hand held, were sectioned with a sharp razor blade. The resulting sections revealed the in situ histoanatomy of the tick. Thin sections of ticks were obtained after either paraffin or polyester wax embedding. Microwave fixation combined with polyester wax embedding made serial thin sections of the different stages of Boophilus ticks possible. The technique preserved antigens as demonstrated by the immunostaining of lymphocytes and erythrocytes infected with Babesia microti in mouse tissues subjected to the same treatment as the ticks. With the microwave fixation-polyester wax technique, the specimen preparation time from fixation to the section on the glass slide was reduced to less than 8 h.     

Ultrastructural heterogeneity of gangliogliomas

Gangliogliomas are rare brain tumors, composed of neuronal and glial cells mixed in a different proportion. The basic histopathological pattern of gangliogliomas is well recognized but the variable microscopic appearance still can pose a challenge to the neuropathologist. The authors reanalyzed their series of gangliogliomas in the files of two departments of neuropathology. All analyzed tumors fulfilled the WHO histological criteria of ganglioglioma. Seven tumors were examined by electron microscopy. The following ultrastructural features were graded: presence of dense-cored vesicles, synaptic vesicles, synapses and intermediate filaments, abundant basal membranes, dystrophic neurites, autophagic vacuoles, and multivesicular bodies. Most of the neoplastic neurons were large, polyglonal or oval with well-developed subcellular organelles, round nuclei, and prominent nucleoli. In most cases there were abundant dense core vesicles, observed in both the tumor cell bodies as well as in their processes. Synapses were typically observed. Intermediate filaments were abundant in all tumors. The most intriguing ultrastructural finding was abundant presence of autophagic vacuoles. In 4 cases, multivesicular bodies were observed. All of the tumors with multivesicular bodies also contained abundant autophagic vacuoles.     

Multifocal,nascent, and invasive myoepithelial carcinoma (malignant myoepithelioma) of the breast: an immunohistochemical and ultrastructural study

This report describes the light microscopic (LM), immunohistochemical (IHC), and electron microscopic (EM) features of a multifocal, nascent, and invasive myoepithelial carcinoma of the breast. By LM, the spindle cells disclosed fibrillar acidophilic cytoplasm, mild nuclear atypia, and a low mitotic index. Myoepithelial differentiation was established through IHC (single- and double-labeling techniques) and EM: periductal and infiltrating spindle cells coexpressed total muscle actin, alpha-smooth muscle actin, vimentin, cytokeratin 14, and pankeratin, and their EM features were characteristic of myoepithelial cells, i.e., perinuclear tonofilaments, subplasmalemmal bundles of microfilaments with dense bodies, intermediate junctions, poorly developed desmosomes, pinocytic vesicles, and fragmented external lamina. No invasive epithelial cells disclosed luminal differentiation (by LM, IHC, EM), identifying, thus, this neoplasm as a pure spindle cell myoepithelial carcinoma of the breast.     

Immunocytochemical study using a GABA antiserum for the demonstration of inhibitory neurons in the rat locus ceruleus

The localization of GABA-like immunoreactivity in the locus ceruleus of rats was studied by the peroxidase-antiperoxidase (PAP) method using a purified antibody raised against GABA applied to paraffin sections, with counterstaining by cresylecht violet, and to floating sections for preembedding immunoelectron microscopy. A few medium-sized and some small neurons showed GABA-like immunoreactivity in both nuclei and perikarya. The preferential localization of these immunopositive neurons in the marginal parts of the locus ceruleus suggests that they are inhibitory local circuit neurons located between this center and the afferent fiber systems. Some of the immunoreactive neurons displayed homogeneous and heterogeneous ( paired cells) patterns. Occurrence of the GABA-GABA interaction is indicated. Immunopositive bouton forms are located close to every positive and negative neuron. Electron microscopy confirms GABA-like immunoreactivity in both medium-sized and small neurons of the locus ceruleus and demonstrates that immunoreactive boutons are axosomatic and axosoma spine symmetric synapses on immunopositive and immunonegative cell bodies. These immunocytochemical results support the existence of inhibitory interneurons in the locus ceruleus.     

Factor VIII-related antigen in malignant hemangioendothelioma of the thyroid: additional evidence for the endothelial origin of this tumor

Malignant hemangioendothelioma of the thyroid gland, which most often originates in a hemorrhagic nodule, is a well-known entity in European alpine regions with endemic goiter. In other parts of the world it very rarely has been diagnosed. This tumor may display considerable morphologic variation and often has been interpreted as a variant of undifferentiated carcinoma. In 13 out of 20 thyroid tumors, classified by light microscopy as malignant hemangioendothelioma, Factor VIII-related antigen, a marker for endothelial cells, was demonstrated in neoplastic cells with the help of immunohistochemical technics applied to conventional paraffin sections. In one case, in which material suitable for electron microscopy was available, Weibel-Palade bodies were found in tumor cells. These findings add strong support to the notion of an endothelial origin of this neoplasm.     

Immunofluorescent characterization of fibronectin, laminin, and keratin in normal and neoplastic human mammary epithelial cells in culture and in breast tissue sections

Employing indirect immunofluorescent staining, primary and secondary serum-free cultures and frozen sections of human mammary tissue, normal and neoplastic, were examined for the presence and distribution of fibronectin (FN) and keratin, and frozen sections also for laminin (LM). The epithelial cell purity of the cultures was confirmed by the observation that all cells stained with anti-keratin antibody. In confluent cultures, FN was absent at the apical cell surface, and was seen as a fibrillar matrix exclusively beneath the epithelial monolayer, at the cell-substratum interface. No differences were noted between normal and neoplastic cells in vitro. In sections of normal breast tissue, FN was localized in the basement membrane zone (BMZ) and in the connective tissue stroma. A distinguishing feature of the neoplastic tissue was the considerably more intensive anti-FN immunofluorescence of the stroma. In normal tissue sections, LM was present exclusively in the BMZ, where it formed a continuous, well-delineated, smooth line; this line was found to be distorted, interrupted, and sometimes entirely absent in the neoplastic tissue. The cytoplasm of all cultured cells, neoplastic as well as normal, exhibited a dense network of keratin filaments that was especially prominent around the nucleus. In the sections, keratin was ubiquitously present in the epithelial cells, predominantly along the interior border of the surface membrane; in the neoplastic tissue, this pattern was markedly disorganized, and some of the cells failed to express the substance.     

A modified technique to process peripheral nerves for light microscopy

A new technique is described to fix, paraffin embed and cut routine peripheral nerve biopsies for light microscopy. Buffered glutaraldehyde pH 7.4 is used as a common fixative for both light and electron microscopy. The nerve bits for LM are embedded in paraffin using the 'reverse embedding' method and blocks are trimmed to cut sections on the ultra microtome using glass knives. The uniformly thin sections can be utilised for any special stain and the good resolution of these thin sections facilitates better observation and documentation.