bluuebunny/crossref_metadata_2025 huggingface dataset:
Anomolous staining patterns in immunohistologic studies of malignant lymphoma.
Authors: Isaacson P , Wright D H
Published: April 2011

A number of immunoperoxidase studies of malignant lymphomas have reported polytypic light chain staining of neoplastic cells, thus bringing into question the concept that the monoclonality of B cell lymphomas is reflected in their synthesis of monotypic light chain. In this study of a large number of Ig positive lymphomas, staining for a wide variety of antigens has identified clear differences between monotypic Ig synthesizing cells and cells staining polytypically which appear to be taking up Ig from the environment. Attention to the nature of Ig staining and staining for J chain were the two most useful criteria in differentiating Ig synthesis from uptake. The results confirm that malignant B cell lymphomas synthesise monotypic Ig.
Simultaneous visualization of two antigens in the same tissue section by combining immunoperoxidase with immunofluorescence techniques.
Authors: Lechago J , Sun N C , Weinstein W M
Published: April 2011

A simple method was developed whereby immunoperoxidase and immunofluorescence techniques were applied in consecutive steps to demonstrate the presence of two antigens in the same tissue section. This method was applied in three model, two antigens were shown: a) each (gastrin and pepsinogen II) inside one of two different cell types (gastrin (G) and antral peptic cells), b) each (kappa or gamma light chains) inside different cells of the same type (plasma cells); also, both (kamma and gamma light chains) inside the same cell (Reed-Sternberg cell), and c) both (pepsinogen I and II) inside the same cell (chief cell of oxyntic glands). The results could be viewed and photographed either simultaneously, when the antigens were in different cells, or sequentially, when the antigens were in the same cells.
A quantitative immunoperoxidase procedure employing energy dispersive x-ray analysis.
Authors: Siegesmund K A , Yorde D E , Dragen R
Published: April 2011

A method is described for substituting gold for osmium as a marker in the unlabeled antibody technique. The gold marker can be detected in the light or electron microscope. The gold-labeled reaction product can be detected in lower concentrations than osmium and can be used as the basis for quantitating antigen concentrations in cells and tissues with the scanning electron microscope and energy dispersive x-ray analysis.
Immunocytochemical studies of the evolution of islet hormones.
Authors: Falkmer S
Published: April 2011

By using both immunofluorescence and peroxidase-anti-peroxidase procedures to detect cells producing the four islet hormones, supplemented by biochemical, biological, and radioimmunological assays of tissue extracts, it has been shown that insulin seems to be the most original hormone, apparently occurring already in invertebrates in cells of open type in the alimentary tract mucosa. Insulin cells also predominate in the first islet organ, namely that of the cyclostomes. The order of appearance in the endocrine pancreas during the subsequent evolution is: somatostatin; glucagon; and the pancreatic polypeptide. Even in lower vertebrates pancreatic polypeptide cells occur in those parts of the pancreas situated in close proximity to the gut.
Cytochemical localization of alkaline phosphatase and Na+-pump sites in adult rat colon.
Authors: Vengesa P B , Hopfer U
Published: April 2011

The cellular and subcellular locialization of alkaline and K+-dependent phosphatase activities in the colonic mucosa of adult rats and rabbits was studied with the electron microscope. The 1-cysteine-sensitive alkaline phosphatase activity was observed in the brush border membrane of the chief cells. The contraluminal plasma membrane of chief cells was devoid of this enzyme activity. In contrast, the cardiac glycoside-sensitive K+-dependent phosphatase was predominantly localized in this region of the cheif cells.
A mammary gland whole mount technique that preserves cell fine structure for electron miscroscopy.
Authors: Strum J M
Published: April 2011

A mammary gland whole mount technique has been developed that preserves cell fine structure and makes it possible to also examine the preparations by electron microscopy. The glands are placed on glass microscope slides, fixed in a paraformaldehyde-glutaraldehyde mixture, defatted in acetone, stained with 0.5% methylene blue (or trypan blue) in saline, and dehydrated in ethanol. They are evaluated and photographed in 100% ethanol. Then specific areas (i.e. containing small growths, tumors, or other lesions) are selected, excised and prepared for electroscopy. The ultrastructural preservation is good, organelles are evident and there is no observable dye precipitate. The only unusual finding is that cell membranes display a "negative" image.
Cytochemical localization of adenylate cyclase.
Authors: Cutler L S , Christian C P
Published: April 2011

This study examined 1) the effect of lead and fixation on adenylate cyclase activity, 2) the effect of lead on App(NH)p, and 3) the specificity of App(NH)p as a substrate for adenylate cyclase under the conditions of the cytochemical assay. The results indicated that: 1) fixation that provides adequate structural preservation inhibits enzyme activity to varying degrees depending on the tissue, fixative, length and temperature of fixation; 2) millimolar concentrations of lead do not negatively affect the adenylate cyclase activity of several different tissues (especially if 10 mM NaF is present); 3) lead does not cause the nonenzymatic hydrolysis of App(NH)p; 4) the App(NH)p obtained from the supplier is contaminated and should be purified before use, since lead can interact with the contaminants and this may be a source of error in the assay; and 5) adenylate cyclase appears to be the major enzyme that cleaves App(NH)p under cytochemical conditions.
Reliability and specificity of membrane adenosine triphosphatase localizations.
Authors: Firth J A
Published: April 2011

The main development in ATPase cytochemistry in recent years is the development of three methodologically independent approaches to the localization of Na+, K+-ATPase. These methods in general support one another well and have led to significant insights into the mechanisms of bulk cation transport at several important sites. The area most in need of development is now the problem of specific localization of divalent ion-dependent ATPases at the plasma membrane. The value of the traditional Wachstein-Meisel methods appears to be very limited, and progress will depend on the introduction of more specific techniques.
Autoradiographic localization of tritiated ouabain-sensitive sodium pump sites in ion transporting epithelia.
Authors: Ernst S A , Mills J W
Published: April 2011

The freeze-dry autoradiographic method devised originally by Stirling (J Cell Biol 53:704, 1972) to localize Na+ pump sites with (3H)ouabain is reviewed. Biochemical, physiological, and autoradiographic data are discussed which establish that ouabain binding to intact tissue conforms to rigid criteria for high Na+ pump specificity. Among these are that glycoside binding exhibits saturation kinetics, ligand dependence, and close correlation with degrees of inhibition of Na+-K+-ATPase and Na+ transport. Moreover, localization of Na+ pump sites by this technique shows a cell and membrane specificity which mirrors that obtained by cytochemical and immunocytochemical methods. In addition to resolving cell-specific patterns of localization in heterogeneous tissues, the demonstration of Na+-K+-ATPase by these techniques indicates that Na+ pumps are distributed uniformly along plasmalemmal surfaces and are restricted to the basolateral interface in reabsorptive and secretory epithelia despite the opposing polarity of net transepithelial electrolyte transport.
An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations.
Authors: Takamiya H , Batsford S , Vogt A
Published: April 2011

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.
Surface distribution of monosialoganglioside GM1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study.
Authors: Ackerman G A , Wolken K W , Gelder F B
Published: April 2011

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.
Autoradiographic localization of carbonic anhydrase in the rabbit ciliary body.
Authors: Muther T F , Friedland B R
Published: April 2011

The high binding affinity of acetazolamide for carbonic anhydrase (K1 congruent to 10(-8) M) was employed to demonstrate the distribution of the enzyme in the rabbit ciliary body by incubating the tissue with 3H-acetazolamide (1.5 Ci/mmol). Specificity of binding was ascertained by displacing 3H-acetazolamide with a high concentration of unlabeled ethoxzolamide (K1 congruent to 10(-9) M). Wedges of the globe anterior to the ora serrata were incubated in bicarbonate buffered physiological saline, 95% O2/5% CO2, at 0 degrees C for 2 hr with either 3H-acetazolamide (0.2 microM), 3H-acetazolamide and unlabeled ethoxzolamide (100 microM), or physiological saline alone. They were then washed for 2 hr in fresh physiological saline and processed for autoradiography. The autoradiographs showed the label localized in both pigmented and nonpigmented layers of ciliary body epithelium in the pars plicata and in the iridial processes. The epithelia of both crests and troughs showed localization of label. In contrast, no concentration of label was found in the stroma of the ciliary body, including vascular endothelium, and in the epithelia of the pars plana. In sections that were incubated with 3H-acetazolamide in the presence of an excess of unlabeled ethoxzolamide, no localization of label occurred. These findings suggest that the epithelia of the pars plicata, but not those of the pars plana, contain carbonic anhydrase. This is consistent with hypotheses restricting aqueous humor formation to the pars plicata.
Distribution of H-2 microenvironments in the mouse thymus. Immunoelectron microscopic identification of I-A and H-2K bearing cells.
Authors: Van Ewijk W , Rouse R V , Weissman I L
Published: April 2011

Antigens coded for by the major histocompatibility complex (MHC) are differentially expressed in the mouse thymus. Immunoperoxidase studies of frozen thymus sections incubated with monoclonal (hybridoma) anti-I-Ak antibodies revealed a dendritic straining pattern in the cortex and a confluent staining pattern in the medulla. Serial sections incubated with monoclonal anti-H-2Kk antibodies showed that H-2Kk antigens were only present at detectable levels in the medulla. Microenvironments expressing H-2Kk antigens also expressed I-Ak antigens. In cortico-medullary regions, relatively large MHC-negative areas were found. These areas appeared to connect to perivascular spaces surrounding blood vessels. Using a new postfixation labeling method for the detection of cell surface associated antigens on cells of the lymphoid system in situ, we have characterized the nature of MHC positive cell types at the ultrastructural level. These studies show that epithelial-reticular cells are the major MHC positive elements in the thymus. Lymphocytes in the medulla and in cortico-medullary bounderies are also MHC positive, however, lymphocytes in the cortex were not detectably labeled. These findings support the contention that epithelial-reticular cells are involved in the H2-restriction process during T cell maturation.
Immunocytochemical localization of renin in kidneys and submandibular glands of SWR/J and C57BL/6J mice.
Authors: Tanaka T , Gresik E W , Michelakis A M , Barka T
Published: April 2011

By using antibodies against highly purified submandibular gland renin, renin was localized immunocytochemically at the light and electron microscopic level in the submandibular glands and kidneys of adult male SWR/J and C57BL/6J mice. In accord with the data of Wilson et al. (Proc Natl Acad Sci USA 75:1185, 1977), renin was demonstrable only in the submandibular glands of SWR/J mice (high strain), where it was confined to the secretory granules of the granular convoluted tubules. In the kidneys of both strains, renin was confined to epithelioid cells of the juxtaglomerular apparatus. Electron microscopically immunostaining was restricted to the granules of the juxtaglomerular epitheliod cells. Morphometric analyses suggested that the kidney of the C57BL/6J mice contained more immunoreactive complexes per unit volume of cortex than SWR/J mice kidney. The data indicate that submandibular gland renin cross-reacts with kidney renin, but that genetic controls of these polypeptides in the two organs are independent.
Isotope decay range distribution curves for use in the analysis of electron microscope autoradiographs.
Authors: Blackett N M , Parry D M , Baker J R
Published: April 2011

Analysis of autoradiographs at the electron microscope level requires special procedures, since the size of the radioactive structures visualized are comparable to the range of the radioactive decay particles emitted. Quantitative analysis in these circumstances requires that the sizes, shapes, and juxtaposition of the various structures be taken into account in relation to the range distribution of silver grains, produced by the decays, from a point source for the particular isotope and autoradiographic conditions employed. We present the distribution of silver grains about a point source for the four electron capture isotopes 51Cr, 55Fe, 111In, and 125I. Thin radioactive line sources were constructed and the distribution of autoradiographic grains measured. The grain distributions are discussed in relation to the number of particles per disintegration and their energy and range. A simple calculation enables these line source distributions to be converted into point source distributions, which can then be used for whichever method of quantitative analysis is considered appropriate for a particular problem. An outline is given of some of the more important aspects of various methods of analysis.
Ultrastructural cytochemistry and radioautography of complex carbohydrates in heterophil granulocytes from rabbit bone marrow.
Authors: Parmley R T , Eguchi M , Spicer S S , Alvarez C J , Austin R L
Published: April 2011

The subcellular route of incorporation of complex carbohydrates into rabbit heterophil primary granules and their subsequent intragranular distribution during granule maturation were studied with ultrastructural, cytochemical, and radioautographic methods. High iron diamine (HID) staining of sulfated glycoconjugates in primary granules was partially diminished after treatment with chondroitinase ABC or after removal of N-sulfate groups with nitrous acid, but was not altered by exposure to hyaluronidase, trypsin, or HCl. Subsequent thiocarbohydrazide-silver proteinate (TCH-SP) straining of thin sections increased the density of the HID reaction product. Golgi-derived spherules and very immature morular granules stained weakly with HID-TCH-SP and labeled intensely after a 10 min incubation with 35SO4. After a 60 min 35SO4 pulse and a 60 min chase, an increase in radiolabeling was observed in granules with HID stained, fused morular material, and some labeling was present in more mature rim stained granules. Fully mature granules lacked HID or HID-TCH-SP staining, but contained most of the 35SO4 labels after a 60 min pulse and 18 hr chase in vitro. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining of unosmicated thin sections localized vicinal glycol-containing complex carbohydrates in Golgi-associated small vesicles. These vesicles lacked HID-TCH-SP staining and apparently contained neutral glycoprotein. They frequently bordered, in a rosette arrangement, the immature morular granules, but not the more mature primary granules. The PA-TCH-SP method localized complex carbohydrates in the rim of granules precursors and enclosed a spherule or morula, but failed to stain the sulfate-containing material in the morulas or spherules. PA-TCH-SP reactivity was diffusely distributed in moderately mature granules and was decreased in fully mature granules. These results indicate that heterophil primary granule contain several complex carbohydrates including O-sulfated and N-sulfated glycosaminoglycans, as well as vicinal glycol-containing glycoproteins. These complex carbohydrates are transported to immature primary granules by different Golgi-derived organelles. The complex carbohydrates are subsequently distributed differently within primary granules and become masked to staining as the granule matures.
Beta-endorphin and somatostatin in the pancreatic D-cell colocalization by immunocytochemistry.
Authors: Watkins W B , Bruni J F , Yen S S
Published: April 2011

Utilizing highly specific antisera, beta-endorphin and somatostatin immunoreactivity were identified simultaneously in the D-cell of the pancreas of the rat, guinea pig, and man by the fluorescence-immunocytochemical technique. Our observations are consistent with a modulating role of beta-endorphin either within the D-cell or upon A and B cells, thereby regulating the secretion of insulin and glucagon.
An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations.
Authors: Taylor I W , Milthorpe B K
Published: April 2011

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.
Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry.
Authors: Mesulam M M , Hegarty E , Barbas H , Carson K A , Gower E C , Knapp A G , Moss M B , Mufson E J
Published: April 2011

In experiments that use horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) for tracing neural connections, the activity of tissue-bound enzyme as well as the stability of the resultant reaction product are influenced by the duration of storage, the composition of the storage medium, the type of counterstaining and even the details of histological dehydration. Furthermore, the conditions for preserving HRP activity are very different from those necessary for preserving the stability of the tetramethyl benzidine (TMB) reaction product. Thus, tissue-bound HRP activity is stable at a neutral pH, while a much lower pH, around 3.3, is required for preserving the stability of the TMB reaction product. Recent evidence indicates that the stabilization bath in sodium nitroferricyanide that was previously recommended is not necessary. However, gradual dehydration of mounted sections is essential for long-term stability. Excessive counterstaining and excessive dehydration interfere with the detection of reaction product. These considerations are pertinent to experiments using free HRP as well as to those where the enzyme has been conjugated to wheat germ agglutinin.
Collagen immunotyping in human liver: light and electron microscope study.
Authors: Grimaud J A , Druguet M , Peyrol S , Chevalier O , Herbage D , El Badrawy N
Published: April 2011

Types I, III, IV, and AB collagens have been extracted from human cirrhotic livers and specific antibodies have been raised in rabbits and purified. Histological immunofluorescent staining of collagen types in normal and fibrotic human livers reveals the respective distribution of the various collagens among the hepatic connective matrix and the modification of the normal pattern in fibrosis: types I and III appear to be the main components of the fibrotic connective matrix in enlarged portal spaces and of the Dissian reticulin framework; type IV collagen deposits are thickened around portal vessels and ducts and outline lobular capillarized sinusoids; type AB collagen appears as thin punctual deposits in portal and Dissian fibrotic connective matrix. Ultrastructural immunoperoxidase labeling of type I and III collagen makes it possible to identify the typical collagen fibers, using 65 nm periodicity, as type I collagen and the fibrillar associated network as type III collagen. Fibers of type I collagen are preferentially organized in large dense bundles in Dense Connective Matrix Organization (DCMO), since fibrillar type III collagen network is predominant in Loose Connective Matrix Organization (LCMO) surrounding vascular and biliary tracts.
Immunofluorescence analysis of normal and malignant lymphoid tissues with selected combination of antisera.
Authors: Janossy G , Thomas J A , Habeshaw J A
Published: April 2011

Tissue sections stained with combinations of antisera labeled with different fluorochromes (i.e., conventional antisera to human immunoglobulin classes, T lymphocyte antigens, and Ia-like p28,33 antigens used in various double combinations with each other or with different mouse monoclonal antibodies) allow the identification of the different areas of lymph nodes in serial sections and provide great flexibility as well as precision in the analysis of the distribution and relationship of normal and malignant cells. Lymphoid microenvironments in the thymus and the paracortical areas of lymph nodes are described. The close association of T lymphocytes and nonlymphoid cells expressing large amounts of Ia-like antigens (such as interdigitating reticular cells and endothelium) may be relevant for the understanding of immunoregulatory disorders such as dermatopathic and angioimmunoblastic lymphadenopathies and some malignancies (e.g., mycosis fungoides) were the expression of Ia-like antigens on non-T cells seems to be abnormally abundant. The analysis of immunoglobulin and membrane marker expression of normal and malignant B cells and their relation to T cells can also be related to the histology of the disease. These studies are clinically useful for the classification of childhood lymphomas, the differential diagnosis of anaplastic carcinomas and lymphomas, and in the study of the early stages of lymphomas.
Intracellular localization of fibronectin using immunoperoxidase cytochemistry in light and electron microscopy.
Authors: Hedman K
Published: April 2011

An immunocytochemical staining method for light and electron microscopy was developed to permit adequate penetration of staining conjugates with high specificity, while preserving acceptable ultrastructure. For this purpose an indirect immunoperoxidase method with Staphylococcal protein A-peroxidase conjugates was used in the presence of saponin on aldehyde-saponin-fixed cells. As the first application, fibronectin was localized intracellularly in human embryonic skin fibroblasts. Fibronectin was detected in large amounts in the cisternae of rough endoplasmic reticulum and in 200 nm (secretory?) vesicles. Little fibronectin was present in the Golgi complex; the stacked Golgi cisternae were conspicuously devoid of this protein. The 200 nm vesicles were mostly distributed on the mature side of the Golgi apparatus. These results indicate that fibronectin is exclusively localized to intracellular structures involved in secretory function and suggest that fibronectin may not be processed in significant amounts within the cisternal stacks of the Golgi complex.
Effect of intervening lesions on amino acid distributions in rat olfactory cortex and olfactory bulb.
Authors: Godfrey D A , Ross C D , Carter J A , Lowry O H , Matschinsky F M
Published: April 2011

Levels of the proposed neurotransmitter amino acids glutamate, aspartate, gamma-aminobutyric acid (GABA), and glycine were measured within the layered structures of the olfactory bulb and olfactory cortex following unilateral transections of the lateral olfactory tract or of virtually all fiber tracts of the olfactory peduncle. Distributions of the amino acids on both lesion and control sides were examined and compared by means of a mapping procedure. The results suggest: 1) Glutamate and aspartate are specifically associated with mitral (and presumably also tufted) cell axons and terminals in the piriform cortex. The distribution of aspartate in the olfactory bulb is further suggestive of a specific association of aspartate with mitral cell dendrites and somata. 2) Glutamate might be specifically associated with some centrifugal fibers traveling to the olfactory bulb in or near the anterior commissure. 3) GABA might be specifically related to some certrifugal fibers to the olfactory bulb in addition to its prominent association with granule cells of the bulb. 4) Glycine is unlikely to play a prominent neurotransmitter role in either the olfactory bulb or olfactory cortex.
Immunohistochemical evidence for the intracellular formation of basement membrane collagen (type IV) in developing tissues.
Authors: Laurie G W , Leblond C P , Cournil I , Martin G R
Published: April 2011

Antibodies to type IV collagen obtained from the basement membrane of the mouse EHS tumor were incubated with sections of rat incisor teeth and other tissues for immunostaining by direct or indirect methods. In all locations, the immunostaining was pronounced in basement membranes in which it was restricted to the "basal lamina" layer, from which "bridges" often extended to nearby basal laminae. Usually no immunostaining was detectable in the cells associated with the basement membranes. However, examination of the capillaries at the posterior extremity of the rat incisor tooth, where tissues are at an early stage of development, showed immunostaining not only of the basement membrane, but also of the endothelial cells. The staining was localized in rough endoplasmic reticulum cisternae, some Golgi saccules and their peripheral distensions, and structures believed to be secretory granules. These findings suggest that the synthesis of type IV collagen proceeds along the classical secretory pathways through rough endoplasmic reticulum and Golgi apparatus. At the same time, immunostaining was usually lacking in the cells of the capillaries that had migrated about 2 mm away from the posterior end of the incisor tooth and also in the cells of most other tissues examined, even though the associated basal laminae were reactive. It is, therefore, presumed that the production of type IV collagen may be high in cells at an early stage of development and that any later production and turnover of basement membrane collagen can only be minimal.
Immunocytologic analyses of 10 nm intermediate filaments in the nervous system of Myxicola.
Authors: Eng L F , Lasek R J , Bigbee J W , Eng D L
Published: April 2011

Antibodies prepared in rabbits against Myxicola infundibulum neurofilaments have been employed to stain neurofilaments immunohistochemically in intact Myxicola infundibulum nervous tissue. Paraffin-embedded and frozen sections (5--6 mu) were examined at the light microscopic level with Sternberger's peroxidase-antiperoxidase method, and Vibratome (20--40 mu) sections were studied at the ultrastructural level with Nakane's conjugated peroxidase method. The neurofilament antibody stained only neurons and axons at the light microscopic level. The staining pattern at the electron microscopic level corresponded to the neurofilaments within axons and neurons. Glial cells, which surround the axons, contain large bundles of filaments that resemble astrocytic filaments in mammalian astrocytes. These filaments do not stain with the anti-neurofilament antibody. Neurons, neurofilaments, glial cells, glial filaments, and nonnervous tissue showed no peroxidase staining when specific antiserum absorbed with neurofilaments was used. These structures were also unstained when antiserum to the glial fibrillary acidic protein of mammalian central nervous system astrocytes was substituted for the neurofilament antiserum. Therefore, in Myxicola infundibulum, the antigenic determinants of the neurofilament protein, as recognized immunohistochemically by anti-neurofilament protein antibodies, are not shared with those of glial filaments.
Fibronectin presence in native collagen fibrils of human fibroblasts: immunoperoxidase and immunoferritin localization.
Authors: Furcht L T , Smith D , Wendelschafer-Crabb G , Mosher D F , Foidart J M
Published: April 2011

Fibronectin is a major constituent of the fibroblast extracellular matrix. Fibronectin binds to collagen, mediates fibroblast adhesion to collagen, and is synthesized and secreted into the medium of cultured fibroblasts. Affinity-purified antibodies to fibronectin and collagen were localized using the peroxidase-antiperoxidase method or with ferritin-coupled secondary antibodies. Using human fibroblasts cultured under routine conditions, fibronectin and procollagen I react in a nonperiodic manner with: 1) approximately 10 nm extracellular fibrils, 2) cell membrane, and 3) membrane-associated vesicles. All fibrils react with both antibodies, suggesting some form of codistribution of fibronectin and collagen in these fibrils. Treatment with ascorbate leads to the development of a larger diameter extracellular fibril, approximately 40 nm in diameter. These large diameter fibrils are clearly collagen fibrils as documented by the procollagen antibody reaction. Importantly, fibronectin is bound to or a constituent of these "native" or cellular made collagen fibrils. Fibronectin and procollagen antibodies localized with the peroxidase-antiperoxidase method have a 70 nm axial repeat of reaction product on ascorbate-treated fibroblasts. Localization of antibodies with ferritin-labeled secondary antibodies is less satisfactory, but supports the basic observations made with the unlabeled antibody enzyme method. This observation rules out any potential criticisms. Although it is more difficult to observe with immunoferritin, there is an indication that antibodies to fibronectin react with an axial periodicity on cellular produced collagen fibrils.
Use of a specific antiserum for renin detection in human kidney.
Authors: Camilleri J P , Phat V N , Bariety J , Corvol P , Menard J
Published: April 2011

The use of anti-human renin antibodies made possible the intrarenal localization of renin in human kidney by immunofluorescence. In normal kidney, only some juxtaglomerular apparatus (JGA) were fluorescent. In these JGA, granular or diffuse fluorescence was only seen in afferent arterioles and was not present in all cells. In the ischemic areas of partially infarcted kidney, fluorescence was seen in all JGA and in interlobular arteries. In these arteries the most eccentric cells were often the most positive. In the nonischemic areas of the same kidneys, fluorescence was not seen in JGA, but was observed in proximal tubular cells, suggesting the reabsorption of filtered renin at this site.
Immunocytochemical characterization of ACTH-like immunoreactivity in cerebral nerves and in endocrine cells of the pituitary and gastrointestinal tract by using region-specific antisera.
Authors: Larsson L I
Published: April 2011

The development and use of region-specific antisera for characterizing pituitary and extrapituitary ACTH immunoreactivity are described. The pituitary corticotrophs and melanotrophs, as well as a system of cerebral nerves, contain antigenic determinants, indistinguishable from those of true, pituitary ACTH [1-39]. The distributional patterns of cerebral nerves, most probably containing ACTH [1-39], is of interest in view of documented behavioral effects of ACTH fragments, as well as the possible interaction between ACTH and certain opioid peptides. Studies on antropyloric gastrin cells, previously reported to contain immunoreactive ACTH-like material indicate that the main form of immunoreactive peptide stored in these cells contains only part of the ACTH [1-39] sequence. Its relation to fragments of the ACTH molecule, as well as to yet unknown (hormonal) peptides, is discussed.
5'-Nucleotidase activities in cultured rat liver epithelial and fibroblast cells.
Authors: Berman J J , Tong C , Williams G M
Published: April 2011

Cell cultures of adult rat liver produced two distinct morphologic cell types: epithelial cells polygonal in shape and growing in nests of closely apposed cells, and fibroblast cells stellate in shape with little cell-cell contact at low density growth, but aligning in parallel arrays at high density. These two morphologic variants displayed dramatic differences in histochemically demonstrable 5'-nucleotidase activities. Fibroblast cells exhibited great activity throughout the cytoplasm with no concentration of activity in the cell membrane. The lesser activity in epithelial cells was concentrated on the cell membrane. The importance of this finding to the interpretation of data derived from experiments with whole liver homogenates is discussed.
Simplified in situ preparation of cultured cell monolayers for electron microscopy.
Authors: Kawamoto K , Hirano A , Herz F
Published: April 2011

The use of xylene for the easy separation of cultured cells embedded in situ from their plastic growth surface is described. This step simplifies the preparation of cell monolayers for electron microscopy.
Acid alpha-naphthyl acetate esterase activity in stored buffy coat smears.
Authors: Giorno R , Beverly S
Published: April 2011

The stability of acid alpha-naphthyl acetate esterase activity in smears of buffy coats was studied. Fixed smears may be stored up to 20 days at 25 degrees C. Unfixed smears may be stored up to 24 hr at 25 degrees C before there is a significant decrease in activity. In constant, fixed or unfixed smears held at -10 degrees C begin to lose activity within 24 hr.
An improved method for the collection of serial cells for electron microscopic analysis of their immunocytochemical stain.
Authors: Ellison D G , Childs G V
Published: April 2011

A technique to find serial views of cells stained for more than one hormone is described. As the number of stains employed is increased, there is less probability that matching views for all stains will be found. We have found a set of 6 series (including 2 sections per grid and 3 alternating stains) to be optimal for recognition of morphology and collection of a significant number of cells.
Reaction of oxytalan fibers in human periodontium to mechanical stress. A combined histochemical and morphometric analysis.
Authors: Jonas I E , Riede U N
Published: April 2011

A combined histochemical and morphometric analysis was performed on normal and mechanically stressed periodontal tissue obtained from upper first premolars of ten boys. Oxytalan fibers were demonstrated histochemically according to the method of Fullmer and Lillie. Quantitative and qualitative oxytalan fiber alterations were evaluated by a morphometric analysis. The morphometric results proved that the oxytalan fiber reaction to increased mechanical stress was fibroplasia, fiber lengthening, and narrowing of the fiber diameter. No differences existed between the oxytalan fiber reaction in tension and pressure zones. These findings imply that the processes of functionally induced remodeling are not the same in the soft and hard tissue.
Differences between the O-acetylated sialic acids of the epithelial mucins of human colonic tumors and normal controls: a correlative chemical and histochemical study.
Authors: Reid P E , Culling C F , Dunn W L , Ramey C W , Magil A B , Clay M G
Published: April 2011

Chemical analyses, together with histochemical assessments, were carried out on specimens of adenocarcinoma of the colon and histologically normal colonic epithelium (from resection margins from cases of carcinoma of the colon). In the epithelial glycoproteins of the normal tissue, both chemical and histochemical investigations indicated that the great majority of the sialic acids contained a side-chain O-acyl substituent located at position C8, whereas the side-chain substitution of the sialic acids of tumor glycoproteins was markedly reduced. Chemical analysis of the normal glycoproteins indicated that the great majority of the sialic acids were resistant to digestion with Vibrio cholerae neuraminidase, presumably due to an ester substituent at C4. The sialic acids of the tumor glycoproteins were significantly different from normal, in that they were less resistant to digestion with neuraminidase (p greater than 0.01), and therefore had a lower percentage of substitution at C4 (p greater than 0.01).
Thyrotropin releasing hormone, somatostatin, and enkephalin: distribution studies using immunohistochemical techniques.
Authors: Johansson O , Hökfelt T
Published: April 2011

Using the indirect immunofluorescence technique of Coons and collaborators, the localization of thyrotropin releasing hormone, somatostatin, and enkephalin was studied in the rat central nervous system. These three peptides were found in neurons throughout the brain and spinal cord; the latter two peptides were also found in the peripheral nervous system and in certain endocrine cells. Utilizing the peroxidase--antiperoxidase technique of Sternberger and collaborators the neuropeptides were also traced at the ultrastructural level. They seemed to be mainly confined to so-called large dense core vesicles (diameter around 1,000 A), localized within cell bodies and nerve terminals.
A modified peroxidase--antiperoxidase procedure for improved localization of tissue antigens: localization of substance P in rat spinal cord.
Authors: Vacca L L , Abrahams S J , Naftchi N E
Published: April 2011

A procedure is presented which modifies the Sternberger peroxidase--antiperoxidase (PAP) technique in order to visualize additional amounts of immunodeposits representing the antigen substance (SP) in 5-micrometer paraffin tissue sections of rat spinal cord. For increased sensitivity, the new procedure utilizes a "double bridge" and diaminobenzidine in low pH buffer. The modifications have made possible the visualization of immunoreactive beaded processes and punctate bodies, which were then traced to determine patterns of SP circuitry. Using the modified PAP procedure, the greatest number of immunoreactive processes appeared in the dorsal horn, where some punctate bodies and varicose processes could be seen adjacent to the myelinated afferent fiber bundles that penetrate the substantia gelatinosa as dorsal root collaterals. Additional immunoreactive processes and punctate bodies coursed through the myelinated afferent fiber bundles that penetrate the dorsolateral white matter, and extend into the intermediolateral gray region. Substance P was also identified within immunoreactive processes found in Rexed's laminae V and VI, as well as the central canal region, the dorsal gray commissure, and the ventral gray and white commissures. Since the modifications improved the visualization of SP-containing processes in sparsely populated regions of the spinal cord, especially the ventral horn, they may be useful in demonstrating other antigens that normally occur in small quantities within tissues.
Immunohistochemical studies of peptidergic neurons in the dorsal horn of the spinal cord.
Authors: Seybold V , Elde R
Published: April 2011

The indirect immunofluorescence technique was used to localize substance P, somatostatin, methionine--enkephalin, neurotensin, and oxytocin in the dorsal horn of the rat spinal cord. The unique distribution of each peptide is described and the relative amount of each peptide in laminae I--III of the dorsal horn and the dorsal part of the lateral funniculus qualitatively assessed. Colchicine treatment and dorsal rhizotomy were used to determine, in part, the origin of immunoreactive fibers and terminals observed in the dorsal horn.
Electrolytic degradation of DNA fluorochromes during flow cytometric measurement of electronic cell volume.
Authors: Alabaster O , Glaubiger D L , Hamilton V T , Bentley S A , Shackney S E , Skramstad K S , Chen R F
Published: April 2011

Changes in flow cytometric measurement of DNA content can result from electrolytic chemical degradation of mithramycin, ethidium bromide, and propidium iodide during simultaneous measurement of electronic cell volume. Bench electrolysis also degrades these fluorochromes without changing the quantum yields, even when they are complexed to DNA. In the flow cytometer, electrolytic production of chlorine at the anode is the probable cause of this degradation, since exposure of these fluorochromes to chlorine gas produces the same effect. It is therefore advisable to measure the DNA content distribution alone before simultaneously measuring the DNA content and the electronic cell volume. If unavoidable effects on the DNA distribution are present, narrow forward-angle light scatter should be used as the cell size indicator during dual parameter measurements. Modifying instrument design by reversing electrode polarity might eliminate this problem.
Fluorescent staining of microfilaments with heavy meromyosin labeled with N-(7-dimethylamino-4-methylcoumarinyl) maleimide.
Authors: Namihisa T , Tamura K , Saifuku K , Imanari H , Kuroda H , Kanaoka Y , Okamoto Y , Sekine T
Published: April 2011

For fluorescent staining of microfilaments in cells, heavy meromyosin (HMM) or subfragment-1 (S-1) was labeled with a novel thiol-directed fluorescent dye, N-(7-dimethylamino-4-methylcoumarinyl) maleimide (DACM), instead of the usual dyes, such as fluorescein-isothiocyanate (FITC). DACM-labeled HMM or S-1 gave characteristic fluorescence patterns to a variety of cell types similar to those reported with the use of FITC-labeled HMM or S-1 or with immunofluorescence techniques using anti-actin antibody. The fluorescence of DACM was fairly photoresistant as compared with FITC, so that HMM or S-1 required only 1 mol of the dye per myosin head. Consequently, F-actin need not be used to preserve the actin binding activity of the myosin fragments when labeling with the dye.
Immunocytochemical demonstration of vasopressin and oxytocin in the rat brain by light and electron microscopy.
Authors: Buijs R M
Published: April 2011

Vasopressin and oxytocin were demonstrated by means of the unlabeled antibody enzyme method in the rat central nervous system. Vasopressin and oxytocin fibers of the magnocellular nuclei were found to run towards the amygdala, hippocampus, and spinal cord. From the parvocellular suprachiasmatic nucleus only vasopressin containing fibers were found to run towards the organum vasculosum lamina terminalis, lateral septum, and lateral habenular nucleus. Immunoelectron microscopy, using the preembedding staining technique, frequently demonstrated vasopressin-containing synapses on neuronal structures in the lateral septum, lateral habenular nucleus, and amygdala. These results suggest a putative neurotransmitter role for these neuropeptides.
Improved visualization of luteinizing hormone releasing hormone pathways by immunocytochemical staining of thick vibratome sections.
Authors: Burchanowski B J , Sternberger L A
Published: April 2011

Using 100-micron thick Vibratome sections and a modification of the peroxidase--antiperoxidase method of immunocytochemical staining we achieve a Golgi-like image of luteinizing hormone releasing hormone (LHRH) cells and fibers in mouse brain. Five LHRH pathways are described: 1) A dense projection of fibers from LHRH cells in the medial preoptic and septal areas to the wall of the third ventricle; 2) a projection of fibers from neurons in the bed nucleus of the stria terminalis and the nucleus of the anterior commissure to the subfornical organ; 3) projections of fibers from neurons in the medial septal nucleus and the diagonal band of Broca to the olfactory bulb; 4) fibers which travel within or just lateral to the wall of the third ventricle from the organum vasculosum laminae terminalis to the median eminence; 5) cells and fibers located just dorsal to the optic tracts which project rostrally to the preoptic area and caudally to the level of the median eminence where they course medially to converge and enter the median eminence.