bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde.
Authors: Leist D P , Nettleton G S , Feldhoff R C
Published: April 2011

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.
Comparative analysis of surface membrane immunoglobulin determination by flow cytometry and fluorescence microscopy.
Authors: Gebhard D F , Mittelman A , Cirrincione C , Thaler H T , Koziner B
Published: April 2011

The analysis of membrane surface immunoglobulin (SmIg) on B lymphocytes was carried out in 59 normal individuals and nine patients with B-cell non-Hodgkin's lymphomas by conventional immunofluorescence microscopy and flow cytometry. Five channel settings of a cytofluorograph were evaluated (100, 150, 200, 250, 300) and the mean and standard deviation of the percent positive cells were calculated and compared to the mean and standard deviation of the microscope reading. On the basis of the relative fluorescence reactivity, we were able to determine a fluorescence intensity at which the results of flow cytometry and fluorescence microscopy were comparable. In normal individuals, for cells expressing surface Ia, the channel giving similar results to that of fluorescence microscopy was 150; for kappa and lambda chains, channel 200; for Fab'PV, channel 200; and for IgM, channel 250. In patients with B-cell non-Hodgkin's lymphomas, for cells expressing surface Ia the channel giving similar results to that of fluorescence microscopy was 100; for kappa, channel 100; for lambda, channel 200; for Fab'FV, channel 150; and for IgM, channel 150. Flow cytometric analysis of SmIg appears to be superior to fluorescence microscopy in efficiency, and has the added advantages of being a rapid, sensitive, and objectively quantitative methodology.
Lectin histochemistry of glycolipid storage diseases on frozen and paraffin-embedded tissue sections.
Authors: Alroy J , Ucci A A , Goyal V , Woods W
Published: April 2011

Lectin histochemical studies were performed on frozen and paraffin-embedded brain tissue sections from six cases of galactosylceramide lipidosis (i.e., globoid cell leukodystrophy, or Krabbe's disease) in Twitcher mice and one case of canine infantile GM1-gangliosidosis. The globoid cells in Krabbe's disease stained with Ricinus communis agglutinin-I (RCA-I), peanut agglutinin (PNA), and Bandeirea simplicifolia agglutinin-I (BS-I) in frozen sections. However, paraffin sections and frozen sections pretreated with chloroform-methanol or xylene, from the same animals, stained with Concanavlia ensiformis agglutinin (ConA), wheat germ agglutinin (WGA), and succinylated-WGA (S-WGA), in addition to staining with RCA-I, PNA, and BS-I. The affected neurons of canine infantile GM1-gangliosidosis stained only with RCA-I in frozen sections. In paraffin sections, however, these cells were negative with RCA-I but positive with BS-I, ConA, Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Ulex europaeus agglutinin (UEA-I) in paraffin sections. These results indicate that in paraffin processing of glycolipid storage disease tissue, some lectin receptors are lost and others are unmasked. The retained receptors can be stained with specific lectins and could serve as markers to characterize and differentiate among the various glycolipid storage diseases.
Heterogeneity of basement membranes of the human genitourinary tract revealed by sequential immunofluorescence staining with monoclonal antibodies to laminin.
Authors: Leu F J , Engvall E , Damjanov I
Published: April 2011

We used monoclonal antibodies specific for human laminin to analyze immunohistochemically the heterogeneity of the basement membranes in various parts of the genitourinary tract. By indirect immunofluorescence microscopy we show that antibody 3H11 reacts with all epithelial basement membranes in the kidneys, testes, epididymis, prostate, uterus, oviduct, and ovary, as well as the smooth muscle cells, blood vessels, and nerves. Antibody 4E10 reacted with most epithelial basement membranes in these organs but was unreactive with the basement membranes of peripheral glomerular capillary loops and the basement membranes of the oviductal mucosa, seminiferous tubules, straight tubules, and rete testis. Hilar seminiferous tubules were reactive with 4E10. In contrast to 3H11, which reacted with all vascular, subendothelial, and muscular basement membranes, 4E10 reacted only with the subendothelial basement membrane of capillaries and veins. The difference in the distribution of epitopes could be demonstrated in tissue sections sequentially reacted with two monoclonal antibodies, but only if the antibody of restricted reactivity (4E10) was used first. These data show that the heterogeneous expression of distinct epitopes of laminin in basement membranes can be demonstrated in the same tissue section by sequential staining. This heterogeneity of basement membranes most likely reflects conformational differences in the expression of epitopes on the laminin molecule in various anatomic structures.
Alpha-fodrin in the adrenal gland: localization by immunoelectron microscopy.
Authors: Langley O K , Perrin D , Aunis D
Published: April 2011

Localization of fodrin, the brain equivalent of spectrin (a protein constituent of the erythrocyte membrane cytoskeleton), was investigated at the ultrastructural level in rat adrenal gland. By use of an affinity purified antibody directed against the alpha-fodrin subunit, all chromaffin cells, cortical cells, nerve fibers, and their surrounding Schwann cells were found to be labeled close to the cytoplasmic side of their plasma membranes. The labeling appeared more intense for chromaffin cells, and secretory granules and mitochondria were frequently found to be associated with the zone containing alpha-fodrin in these cells. The immunostained zone was estimated to extend 230 +/- 70 nm into the cytoplasm. This localization is discussed in terms of what is known of the properties of spectrin, and possible roles of the molecule in the chromaffin cell are suggested.
Modification of the protein A-gold immunocytochemical technique for the enhancement of its efficiency.
Authors: Bendayan M , Duhr M A
Published: April 2011

A modification in the protein A-gold immunocytochemical technique has been introduced for amplification of the labeling. This modification consists of performing additional incubation steps with an anti-protein A antibody and the protein A-gold complex. The original antigen-antibody-protein A-gold complex was further incubated with an antibody directed against protein A and then, in a fourth step, again with protein A-gold. This multiple-step protocol results in significant enhancement of the original signal. The modified technique can be applied to either light or electron microscopy protein A-gold immunocytochemistry. The advantage of such an approach is double: it allows for either amplification of the labeling when the original signal is of low intensity or use of highly diluted antibody solutions. The modification introduced was thus found to significantly enhance the efficiency of the technique.
Calmodulin: localization in plant tissues.
Authors: Lin C T , Sun D , Song G X , Wu J Y
Published: April 2011

Calmodulin was purified from bovine brain by preparative SDS-polyacrylamide gel electrophoresis. The denatured, purified calmodulin was used to immunize rabbits to produce antiserum. This antiserum was used to study the distribution of calmodulin in plant tissues by indirect immunohistochemistry. The root tips from corn seeds, oat seeds, peanuts, spaghetti squash seeds, and the terminal buds of spinach were investigated. A method for plant tissue sectioning and inhibition of endogenous peroxide activity was developed. In the corn root section, reaction product from anti-calmodulin was found mainly in the root cap cells. Lesser but significant amounts of calmodulin were localized in metaxylem elements, in some stele cells surrounding metaxylem elements, in apical initials, and in the cortical cells. Similar findings were also observed in other root tips from oat seeds, peanuts, and spaghetti squash seeds. In the terminal buds of the spinach, calmodulin-stained cells were highly concentrated in the apical meristem and leaf primordium. These findings suggest that the high concentration of calmodulin in the root cap may be important in relation to gravitropism and growth development.
Growth patterns and histochemical characterization of bovine mammary corpora amylacea.
Authors: Sordillo L M , Nickerson S C
Published: April 2011

Corpora amylacea in bovine mammary tissue were quantified across a range of size differentials for histochemical properties, lactation age, and lactation stage, in an attempt to characterize amyloid nucleation and growth. At all size classifications, corpora stained positively for amyloid, calcium deposits, and glycoprotein, while staining negatively for mucopolysaccharides. Prevalence of corpora amylacea among the size differentials was unrelated to age of lactating animals, although no corpora were observed in quarters of primiparous heifers at parturition. Corpora amylacea were most abundant during the later stages of lactation for all size differentials, and least abundant during late involution and early lactation. The majority of corpora were observed in alveolar lumens at all stages of lactation. Our results suggest that corpora amylacea development is not restricted to a particular stage of lactation, although their nucleation appears to occur within the alveolar lumens. Gradual increases in both size and numbers of corpora from parturition to late lactation suggest that development of these structures accelerates as lactation progresses. Morphological relationships between corpora amylacea and mammary parenchymal tissue during the later stages of lactation suggest that these structures may have a role in the involutionary process. These findings provide the foundation for additional immunocytochemical techniques to determine the origin of amyloid fibril components.
Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens.
Authors: Pickel V M , Chan J , Milner T A
Published: April 2011

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.
Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.
Authors: Howe W E , Klier F G , Oshima R G
Published: April 2011

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.
Expression of sialic acid on the alveolar surface of adult and fetal human lungs.
Authors: Faraggiana T , Villari D , Jagirdar J , Patil J
Published: April 2011

Lung alveoli are coated by a thin layer of extracellular material rich in anionic charges. The nature of this acid layer and its relationship to the phospholipid surfactant are not known. We investigated the possible presence of sialic acid groups by light and electron microscopy in tissues from normal fetal and adult lungs, using neuraminidase treatment followed by staining with the galactose-binding lectin from peanut, labeled with peroxidase. Our results showed that adult lung does not bear peanut lectin-reactive sites but that a very thin and distinct reactive layer becomes evident after neuraminidase treatment, especially on type II pneumocytes. In fetal lung, the entire surface of the developing respiratory tree is outlined by a strongly peanut lectin-reactive layer even if neuraminidase digestion is not performed. We conclude that the acid coat of the alveolar lining is in part composed of sialic acid residues and that sialic acid is added to the fetal lung as the alveoli mature.
Two embryonic, tissue-specific molecules identified by a double-label immunofluorescence technique for monoclonal antibodies.
Authors: Wessel G M , McClay D R
Published: April 2011

We identify two tissue-specific molecules in the sea urchin embryo by an immunofluorescence technique capable of co-localizing monoclonal antibodies on the same tissue section. The technique uses monovalent Fab-fluorochrome conjugates as secondary reagents to avoid cross-talk of subsequent antibody probes. Using this technique, we show that two cell surface molecules are expressed by different cell populations in the embryo. The technique is generally applicable for antibodies regardless of species or subtype specificity, and uses commercially available reagents. The technique provides sufficient amplification and resolution for analytical work, yet is rapid enough for screening procedures. As a fluorescent counterstain, use of the dye 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) in the protocol provides a distinct fluorescent background staining of the tissue without interference with the specific antibody staining.
Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody.
Authors: Morstyn G , Pyke K , Gardner J , Ashcroft R , de Fazio A , Bhathal P
Published: April 2011

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.
Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells.
Authors: Nagle R B , Böcker W , Davis J R , Heid H W , Kaufmann M , Lucas D O , Jarasch E D
Published: April 2011

Two monoclonal antibodies, KA 1 and KA 4, raised against human epidermis, were biochemically and immunologically characterized and were shown to react with specific cytokeratin polypeptides. On frozen sections of human mammary gland, these antibodies distinguish between myoepithelial and luminal epithelial cells. We present evidence that in these cells KA 1 antibody recognized cytokeratin 5 and KA 4 antibody cytokeratin 19. In normal mammary tissue, KA 4 antibody invariably reacted with the epithelial cells lining the lumina of acini, ductules, ducts, and sinus. In contrast, KA 1 antibody decorated only the myoepithelial and basal epithelial cells of acini, ducts, and sinus. In ductules, however, KA 1 also stained the luminal cells. All 73 invasive lobular and ductal carcinomas studied reacted with KA 4 antibody; five of these were also positive, apparently in the same tumor cells, with KA 1. The tumor cells of in situ carcinomas were also stained in a homogeneous pattern with KA 4 antibody; KA 1 antibody reacted only with the surrounding myoepithelium. In epithelial hyperplasias, the proliferating cells were decorated by KA 1 and KA 4 antibodies in a heterogeneous pattern. Other antibodies were used for comparison. The results are discussed with respect to epithelial differentiation and pathogenesis and to the application of such antibodies for immunohistodiagnosis of mammary lesions.
Reticular fibroblasts in peripheral lymphoid organs identified by a monoclonal antibody.
Authors: Van Vliet E , Melis M , Foidart J M , Van Ewijk W
Published: April 2011

We have produced a panel of monoclonal antibodies directed against nonlymphoid cells in central and peripheral lymphoid organs. In this paper we present the reactivity of one of these antibodies, ER-TR7. This antibody detects reticular fibroblasts, which constitute the cellular framework of lymphoid and nonlymphoid organs and their products. In frozen sections of the spleen incubated with this antibody, the red pulp and white pulp are clearly delineated. Furthermore, the major white pulp compartments--the follicles and periarteriolar lymphoid sheath as well as the marginal zone--are recognized by their characteristic labeling patterns. In lymph nodes, the capsule, sinuses, follicles, paracortex, and medullary cords are clearly delineated. In the thymus and bone marrow no such specialized compartments were demonstrated. ER-TR7 reacts with an intracellular component of fibroblasts. Since ER-TR7 does not react with purified laminin, collagen types I-V, fibronectin, heparan sulfate proteoglycan, entactin, or nidogen, it detects a hitherto uncharacterized antigen. The possible role of the ER-TR7 positive reticular fibroblasts in the cellular organization of peripheral lymphoid organs will be discussed.
Absence of relaxin immunostaining in the male reproductive tracts of the rat and mouse.
Authors: Anderson M B , Collado-Torres M , Vaupel M R
Published: April 2011

By use of the biotin-avidin immunohistochemical method and a homologous antiserum as the primary antiserum, relaxin immunostaining was absent in the testes, prostate, seminal vesicles, and epididymides of the rat. Relaxin immunostaining was also lacking when anti-porcine relaxin serum was employed as the primary antiserum. Furthermore, immunohistochemical studies for relaxin localization in the reproductive tract of the male mouse using both anti-rat and anti-porcine relaxin sera also revealed an absence of the hormone in the reproductive system of this species. Although this study suggests that immunoreactive relaxin is absent in the male reproductive tracts of both the rat and mouse, it raises some questions concerning the reports in the literature of the presence of relaxin-like substances in the male reproductive tracts of other species. These reports are discussed in relation to our current results.
Production and characterization of a monoclonal antibody to human type III collagen.
Authors: Macarak E J , Howard P S , Lally E T
Published: April 2011

Human type III collagen from placenta was isolated and purified for use as an immunogen. A monoclonal antibody was produced which specifically recognizes epitopes unique to type III collagen. The specificity of the antibody was determined by inhibition ELISA, an immunoblot assay, and by immunoprecipitation. Results indicated that the monoclonal antibody recognized only the alpha 1(III) polypeptide chains and did not crossreact with type I, IV, or V collagen. The monoclonal antibody was also used for immunohistochemical localization of type III collagen in tissue sections of human placenta, bovine spleen, and lymph node. In placenta, both large and small blood vessels showed pronounced staining of the tunica media, which contains largely smooth muscle cells, known to synthesize type III collagen. In contrast, the intimal areas and endothelial cells showed no staining with the antibody. In the placental villi, staining was limited to the villous core, where fine fibrillar structures showed strong staining. In lymph nodes, the capsule and pericapsular adipose cells were surrounded by a covering of type III collagen. Within the parenchyma of the node, staining was localized to a branching, reticular array of fine fibers. In the spleen, staining was pronounced in the capsule, splenic trabeculae, and white pulp, where blood vessel staining was especially prominent. The red pulp and splenic sinuses contain little or no type III collagen. The fine network-like or reticular staining pattern found in the lymph node parenchyma is consistent with the staining pattern of the protein reticulin, and suggests that type III collagen may be closely associated with reticulin in certain tissues. Since the role of type III in tissues is unclear, this reagent will be useful in providing new information in this regard.
Immunocytochemical investigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human.
Authors: Voss B , Glössl J , Cully Z , Kresse H
Published: April 2011

Polyclonal antibodies against the core protein of the small chondroitin sulfate-dermatan sulfate proteoglycan from human skin fibroblast secretions were used, after affinity-purification, as a probe to study localization of crossreactive material in several human tissues by indirect immunocytochemistry. In contrast to skin, kidney, and the adventitial layer of aorta, positive staining of brain, liver, cartilage, and intimal and medial layers of aorta required pre-treatment of tissue sections with chondroitin ABC lyase. In all tissues investigated, antigenic material was present in the interstitial space. Filamentous structures were perpendicularly oriented towards basement membranes. In liver, specific staining was seen along the sinusoidal walls. Reticular fibers with or without focal condensations were seen in cerebral cortex and cerebellum. The results suggest a role of small chondroitin sulfate-dermatan sulfate proteoglycan in cell-matrix interactions.
Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin.
Authors: Sonnenberg A , Daams H , Van der Valk M A , Hilkens J , Hilgers J
Published: April 2011

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.
Detection of polymorphic rat MHC antigen determinants in cryostat sections. A method for selecting monoclonal alloantibodies.
Authors: Steiniger B , Klempnauer J , Günther E , Wonigeit K
Published: April 2011

An immunohistological method for detection of rat MHC antigens by unconjugated primary alloantibodies is described. The procedure is based on secondary reagents specific for an allotypic marker of rat kappa light chains and makes possible differentiation between specifically bound alloantibody and the ubiquitously distributed interstitial immunoglobulin in the section. This technique can be used to pre-select monoclonal antibodies to be coupled to biotin or enzymes for histological application.
Phosphatase localization in the endomembrane system of the dinoflagellate Crypthecodinium cohnii.
Authors: Barlow S B , Triemer R E
Published: April 2011

The distribution of four enzymes within the endomembrane system of the protist Crypthecodinium cohnii has been determined using cytochemical localizations with lead as a capture agent. Nucleoside diphosphatase (NDPase) activity, using inosine diphosphate (IDP) and thiamine pyrophosphate (TPP) as substrates, was observed in the Golgi apparatus, with a gradient of increasing reaction product noted in some cells from the cis to trans cisternae. Tubules and vesicles associated with the trans cisternae also contained reaction product. The endoplasmic reticulum exhibited a high activity of glucose-6-phosphatase [with glucose-6-phosphate (G-6-P) as substrate]. Traces of reaction product were also observed in the cis-most and trans-most cisternae of the dictyosomes. Activity of acid phosphatase (AcPase) was observed in Golgi cisternae as well as in associated cytoplasmic vesicles. Heaviest deposition was localized in medial and trans dictyosome cisternae. The cytoplasmic system of flattened vesicles subtending the surface membranes in these cells did not exhibit reactivity with any of the substrates used. The distribution of these enzymes in this algal cell appears similar to that observed in animal cells and suggests that these enzymes may represent markers for algal cell endomembrane compartments.
Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix.
Authors: Tischler A S , Mobtaker H , Mann K , Nunnemacher G , Jason W J , Dayal Y , Delellis R A , Adelman L , Wolfe H J
Published: April 2011

Anti-lymphocyte monoclonal antibody HNK-1 (Leu-7) reacts with the cell surfaces of natural killer (NK) lymphocytes and with myelin-associated glycoprotein (MAG). This antibody reacts intensely with normal and neoplastic adrenal medullary cells. A small proportion of normal pancreatic islet cells, anterior pituitary, and gastroenteropancreatic endocrine cells also show Leu-7 immunoreactivity. In adrenal medulla, ultrastructural immunocytochemical studies and immunoblot analyses reveal that Leu-7 reacts with an intracellular protein of MW 75 KD which is localized within the matrices of the chromaffin granules. The MW of this protein differs from those of MAG and chromogranin A. The findings suggest that Leu-7 immunoreactivity might be a new marker for specific subsets of secretory granules.
Lectin-binding sites in human parathyroid tissue.
Authors: Thiele J , Vierbuchen M , Arnold G , Walgenbach S , Fischer R
Published: April 2011

The aim of this study was to demonstrate several lectin-binding sites in human parathyroid tissue and to correlate these results with functional activity. The following lectins were tested for binding sites with certain carbohydrates (in parentheses): Arachis hypogea (PNA) (galactose), Ulex europaeus I (UEA) (fucose) and concanavalin A (ConA) (mannose). In addition to normal parathyroids used as controls (13 cases), we examined adenomas associated with a clinical picture of primary hyperparathyroidism of differing severity (31 cases), atrophic glands contralateral to a hyperfunctioning adenoma (7 cases), and secondary (renal) hyperplasia (12 cases). Use of PNA (with and without neuraminidase treatment) and UEA yielded negative staining in normal glands, a wide variety of reactions in adenomas, and frequent dense precipitates in atrophic parathyroids, whereas ConA yielded positive staining in all kinds of parathyroid tissue. Assessment of functional activity of adenomas by clinical parameters (pre-operative serum levels of calcium and parathormone) displayed a significant correlation with the semiquantitative grading of the histochemical reactions after PNA and UEA. Lectin-binding sites in parathyroid chief cells of adenomas are believed to indicate some of the cell structures or products directly involved in the secretory process, including degradation. Although ConA may recognize constituent parathyroid glycoproteins, the binding sites for PNA and UEA are thought to be partially associated with secretory glycoprotein (SP-I), as is known from animal experiments. The positive reaction of the atrophic gland may result from degradation enforced by exposure of primarily non-terminal carbohydrate components.
Early stages of synthesis of thyroglobulin (Tg), thyroxine (T4), and triiodothyronine (T3) in fetal rat thyroid. An immunoelectron microscopic study.
Authors: Kawaoi A
Published: April 2011

Immunoelectron microscopy was applied for detection of Tg, T4, and T3 in fetal rat thyroid gland during the early stages of synthesis of these compounds, in order to elucidate the subcellular aspects of onset of their production. Tg was detected in the perinuclear space, rER, and Golgi apparatus as early as the sixteenth day of gestation when no follicles had yet formed. When primitive follicles had formed after the seventeenth day of gestation, both T4 and T3, as well as Tg, appeared on the surface of microvilli and occasionally in their core portions. These findings provide evidence to suggest that follicles are essential to extracellular synthesis of T4 and T3 in fetal rat thyroid gland.
Combined method of PAP immunocytochemistry and autoradiography: application to cell kinetic study in plasma cell dyscrasias.
Authors: Kanoh T , Ohnaka T , Uchino H
Published: April 2011

The plasma cell labeling index (LI), in spite of being a reliable indicator for diagnosis and prognosis of multiple myeloma, has been measured in a limited number of laboratories because of technical difficulties. We have developed a new combined technique, using the peroxidase-antiperoxidase (PAP) method and autoradiography, which has several advantages over previously described methods. The primary advantages of our method in the determination of lymphoid-plasma cell LI% are: (a) no damage to slides during storage of more than 1 year; (b) an exact LI measurement in each morphological variety of pleomorphic immunoglobulin-containing cells; (c) no problem in differentiation of lymphoid plasma cells from early red cell precursors; and (d) a separate LI measurement for those lymphoid-plasma cells composed chiefly, if not exclusively, of monoclonal or neoplastic cells. Because of these advantages, this accurate and less difficult technique will facilitate performance of lymphoid plasma cell LI in a number of laboratories.
Resolution in EM autoradiography V: 45Ca (and 33P).
Authors: Salpeter M M , Nelson R , Harris R , Salpeter E E
Published: April 2011

We devised a specimen to test EM autoradiographic resolution for calcium-45 (and phosphorous-33). A dry emulsion film was used for soluble compounds. We obtained a resolution (HD) value of 2300 +/- 500 A. This value is no larger than that previously obtained for Carbon -14, whose emission is about 1.6-fold lower. This result is as expected on theoretical grounds.
A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites.
Authors: Skutelsky E , Bayer E A
Published: April 2011

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.
An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique.
Authors: van der Loos C M , Das P K , Houthoff H J
Published: April 2011

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).
Effect of tissue fixation on anti-bromodeoxyuridine immunohistochemistry.
Authors: Schutte B , Reynders M M , Bosman F T , Blijham G H
Published: April 2011

We investigated the influence of various fixatives on monoclonal anti-BrdUrd antibody binding of BrdUrd-substituted DNA in tissue sections of routinely processed mouse small intestine after in vivo administration of BrdUrd. For denaturing fixatives such as ethanol or Carnoy's fluid, a standard denaturation protocol showed specific crypt cell labeling. With cross-linking agents such as formalin and glutaraldehyde, a remarkable increase in staining intensity was obtained after tissue digestion with pepsin before acid denaturation. The optimal pepsin concentration was determined for maximal immunoreactivity combined with acceptable morphology.
Demonstration of anionic sites on the luminal and abluminal fronts of endothelial cells with poly-L-lysine-gold complex.
Authors: Vorbrodt A W
Published: April 2011

An attempt was made to demonstrate the anionic sites on the endothelial cell (EC) surfaces of mouse brain micro-blood vessels (MBVs) after embedding of tissue samples in hydrophilic media: Lowicryl K4M, LR White, and Polyamph-10. As a cationic probe, poly-L-lysine-gold complex (PLG), prepared according to the procedure of Skutelsky and Roth (J Histochem Cytochem 34:693, 1986), was used. In ultra-thin sections of brain samples embedded in Lowicryl K4M and LR White, the anionic sites were demonstrated in the entire cross-section of the vessel wall. After embedding in Polyamph-10, however, the anionic sites could not be detected. Brain capillaries, representing blood-brain barrier type MBVs, showed polar distribution of anionic sites, evidenced by more intense labeling of luminal than of abluminal plasma membrane of the EC. Some differences in labeling of ECs and of basement membrane in arterioles and venules were also noted. The use of cationic gold and the ultra-thin sections of tissue samples embedded in hydrophilic media (Lowicryl K4M and LR White) seems to be a promising new method for detection of anionic constituents located on both luminal and abluminal surfaces of the EC, in the basement membrane, and in other components of the vessel wall.
High-resolution localization of acetylcholinesterase at the rat neuromuscular junction.
Authors: Schätz C R , Veh R W
Published: April 2011

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.
Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt.
Authors: Bell P B , Rundquist I , Svensson I , Collins V P
Published: April 2011

We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.
Distribution of Na+,K+-ATPase in rat exocrine pancreas as monitored by K+-NPPase cytochemistry and [3H]-ouabain binding: a plasma membrane protein found primarily to be ductal cell associated.
Authors: Madden M E , Sarras M P
Published: April 2011

We investigated the distribution of Na+,K+-ATPase in rat exocrine pancreas. By use of enzymatic dissociation techniques, pancreatic acini (containing acinar cells and centroacinar ductal cells in a ratio of about 10:1) and all major classes of pancreatic ducts were isolated and analyzed for the presence of Na+,K+-ATPase using K+-NPPase cytochemistry and [3H]-ouabain binding assays. Ultrastructural analysis demonstrated a basolateral localization of ouabain-sensitive enzyme activity in all classes of pancreatic ducts, although the degree of activity varied among the various classes. Qualitative analysis (scale of 0 to + + +) indicated the following enzyme distribution: centroacinar ductal cells (+); intralobular ducts (+ +); interlobular ducts (+ + +); main duct (+ +). In contrast, no reaction product was associated with pancreatic acinar cells even when observed adjacent to enzyme-positive centroacinar ductal cells. Parallel experiments monitoring [3H]-ouabain binding supported the cytochemical studies. When expressed as femtomoles [3H]-ouabain/microgram DNA, the following values were obtained: whole pancreas, 100.3; ducts (pooled intralobular and interlobular), 337.0; acini, 48.2. The acinar value is complicated by the fact that acini contain both acinar and centroacinar cells, but in light of the cytochemical observations we suggest that most of the [3H]-ouabain binding is due to the few ductal cells present in acini. The results suggest that Na+,K+-ATPase is primarily associated with the ductal epithelium of the exocrine pancreas and is differentially distributed among the different classes of ducts.
Use of azide and hydrogen peroxide as an inhibitor for endogenous peroxidase in the immunoperoxidase method.
Authors: Li C Y , Ziesmer S C , Lazcano-Villareal O
Published: April 2011

We describe a simple and effective method for inhibition of endogenous peroxidase activity in the immunoperoxidase technique. Specimens are pre-treated with a mixture of azide and hydrogen peroxide, which is then followed by an indirect immunoperoxidase procedure. Comparison studies showed no significant loss of antigenicity or morphological details by this pre-treatment. The method is most useful for evaluating cell-specific antigens on specimens that have abundant endogenous peroxidase activity, such as blood and bone marrow.
Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells.
Authors: Willingham M C , Richert N D , Cornwell M M , Tsuruo T , Hamada H , Gottesman M M , Pastan I H
Published: April 2011

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.
T-cells in follicular hyperplasia and follicular center-cell lymphomas: an immunoultrastructural study of the T-cell receptor-associated CD3 antigen.
Authors: Angermeier P A , Hartman A L , Swerdlow S H
Published: April 2011

T-cells are an integral part of normal follicular centers and many follicular center-cell (FCC) lymphomas. Because the functional role of these cells remains imprecisely determined and because ultrastructural localization of the T-cell antigen receptor-associated CD3 antigen has not been previously reported, an immunoultrastructural study of four tonsils and four FCC lymphomas was performed using an anti-CD3 antibody (UCHT-1). Normal interfollicular CD3-positive T-cells always demonstrated surface membrane positivity, as did 94% of normal and 88% of neoplastic FCC-associated CD3-positive cells. Conversely, whereas only 5% of normal interfollicular CD3-positive cells showed perinuclear positivity, 58% of normal and 38% of neoplastic FCC-associated CD3-positive cells did (p less than 0.001). Definite endoplasmic reticulum (ER) staining for CD3 was identified in 4% and 8% of cells associated with normal or neoplastic FCC, respectively, but in none of the T-zone lymphocytes. Because the perinuclear space is reported to be a site of protein synthesis in cells with little ER, and because of the occasional ER staining observed in cells with perinuclear staining, perinuclear CD3 positivity probably represents CD3 synthesis in mature tonsillar T-cells. The frequency of perinuclear positivity in the FCC-associated T-cells, together with the loss of surface positivity in some cells at this site, suggests that this could represent in vivo T-cell "activation" in follicular centers, with modulation and resynthesis of the CD3 antigen. Furthermore, the results demonstrate a similar phenomenon in the T-cells associated with neoplastic follicular center cells.