bluuebunny/crossref_metadata_2025 huggingface dataset:
Lysosomes and proteolytic enzyme activities in cultured striated muscle cells
Authors: Bird JW , Roisen FJ , Yorke G , Lee JA , McElligott MA , Triemer DF , St John A
Published: April 2011

Primary cell cultures prepared from chick embryonic skeletal muscle and the rat myogenic line L6 were examined morphologically and biochemically during several stages of development. The L6 cells were cultured to provide three morphologically distinct populations: prefusion, postfusion, and a subclone of cells that did not fuse even at high density. Ultrastructural studies revealed the characteristic morphology of healthy myoblasts. Acridine orange staining and cytochemical localization of acid phosphatase suggest the presence of presumptive lysosomal material. Enzymatic studies of lysosomal cathepsins B, D, H, and L revealed unusually high enzyme specific activities in these homogeneous myoblast populations. No activity was detected for the two nonlysosomal enzymes Ca2+-proteinase and serine proteinase. It is suggested that the lysosomal apparatus and its complement of enzymes play a significant role in the differentiation of muscle myotubes.
Ethidium bromide: a nucleic acid stain for tissue section.
Authors: Franklin W A , Locker J D
Published: April 2011

The phenanthridinium dye, ethidium bromide (EB), selectively intercalates into double-stranded regions of nucleic acids with a large and specific increase in fluorescence. When used for the staining of fixed tissue sections, the dye stains cellular nuclei with excellent resolution of microscopic detail. In some fixed tissues, particularly pancreatic acini, cytoplasm stains intensely and this staining can be abolished by digestion with trypsin and ribonuclease. The orange fluorescence of EB can be easily distinguished from the green fluorescence of fluorescein and EB is thus an excellent counterstain for immunofluorescence. Ethidium bromide is a useful and practical stain for the fluorescence microscopy of tissue sections and, in combination with enzymatic digestion of RNA, provides a simple way to differentially localize DNA and RNA.
Double labeling study of anionic sites and concanavalin A binding sites in monkey macrophages.
Authors: Takata K , Nishiyama F , Hirano H
Published: April 2011

Cationized ferritin (CF) binding, and its effect on the concanavalin A (Con A) binding pattern were studied by the double technique in monkey peritoneal macrophages. CF particles formed clumps and were internalized when cells were incubated at 37 degrees C. Such cells were fixed, and the Con A binding sites were visualized by the Con A-horseradish peroxidase (HRP) method. Using the same specimen, the distribution of CF particles and the Con A-HRP product was observed under an electron microscope. The redistribution and internalization of CF particles did not affect the continuous label of the cell surface Con A binding sites. These observations suggest the independent mobility of cell surface anionic sites and Con A binding sites.
Fundamental aspects of the interaction of propidium diiodide with nuclei acids studied in a model system of polyacrylamide films.
Authors: Tas J , Westerneng G
Published: April 2011

Qualitative and quantitative aspects of the fluorescent propidium diiodide (PI) staining method have been investigated with model films of polyacrylamide gel incorporated with DNA, RNA, and other macromolecular compounds. PI was found to bind specifically to DNA and RNA, most probably by intercalation into double-stranded regions. Proteins, glycogen, and glycosaminoglycans did not show fluorescence after PI staining. Optimal conditions for dye binding and differentiation have been defined. The stability of nucleic acid-PI complexes, as present in model films, was shown to be very high in distilled water, while dissociation rapidly occurred in ionic media. Linear relationships were found between the fluorescence intensity of bound PI and both the thickness of the model films and the amount of DNA or RNA incorporated. The presence of histone protein bound ionically to DNA did not influence the fluorescent PI binding ability in any appreciable amount.
Immunocytochemical demonstration of intracytoplasmic alkaline phosphatase in HeLa TCRC-1 cells.
Authors: Tokumitsu S , Tokumitsu K , Fishman W H
Published: April 2011

The ultrastructural localization of alkaline phosphatase has been examined in cells of a HeLa subline (TCRC-1) that are monophenotypic for Regan isoenzyme expression. Enzyme activity was demonstrated at the cell surface plasma membrane and in certain lysosomes as revealed by the lead citrate method. The regular direct immunoperoxidase procedure utilizing antibodies in IgG or Fab' form showed the same distribution patterns of alkaline phosphatase. However, when the cell surface antigen was blocked in advance with specific unlabeled antibodies and direct immunocytochemistry performed in the presence of saponin, intracellular alkaline phosphatase antigen was observed in the perinuclear space, endoplasmic reticulum, and Golgi apparatus. The results appeared to be concordant with the current concept that membrane glycoproteins are formed in the endoplasmic reticulum, modified in the Golgi apparatus and then transported to the cell surface. Intracellular alkaline phosphatase was observed predominantly in some cell populations especially mitotic cells, suggesting that the enzyme protein was synthesized in and around the mitotic phase. Accordingly, this technique of differential membrane immunocytochemistry appears to provide an opportunity to follow ectopic gene expression as a function of cell cycle and enzyme induction.
HISTOCHEMICAL STUDIES OF THE OLFACTORY MUCOSA OF THE RABBIT
Authors: BURKHARDT BETTY J. , EHRMANTRAUT HARRY C.
Published: April 2011

1. Tests of the olfactory mucosa of rabbits by five different methods indicate that cholinesterase is probably not present in any of the cells of this tissue.

2. Alkaline phosphatase and phosphamidase were not found in the olfactory receptors.

3. Acid phosphatase and sulfhydryl groups were found to be present in the receptors, and the presence of cytochrome oxidase was demonstrated in gross sections of turbinate tissue.
Z- and M-band appearance in different histochemically defined types of human skeletal muscle fibers.
Authors: Sjöström M , Kidman S , Larsén K H , Angquist K A
Published: April 2011

In order to define ultrastructural features, which alone or in combination with other features could be used to identify different types of fibers in human skeletal muscle, frozen biopsy specimens of m. tibialis anterior were serially sectioned. The thawed sections were prepared either for enzyme histochemistry or for electron microscopy. The same fiber was then identified in all serial sections and its ultrastructure examined under the electron microscope. A total of 75 fibers were included in this investigation. Specimens were also conventionally prepared for electron microscopy. Special interest was devoted to the appearance of the sarcomeric Z- and M-bands. In the same fiber, all myofibrils showed the same Z- as well as M-band structure. On the other hand, it was evident that these structures varied from one type of fiber to another in the same muscle and that their appearance were covariant to a great extent. Low level resolution of Type 1 fibers usually showed broad Z- and M-bands with five strong M-bridge lines. In Type 2A fibers intermediate Z-bands were observed. In the middle portion of the M-bands, three strong M-bridge lines were distinct while the two outer lines were relatively weak. Finally, Type 2B fibers usually appeared with narrow Z-bands. The three M-bridge lines in the middle were strong while the two outer ones were very weak, if seen at all. Discriminant analysis showed that about 70% of the fibers should have been correctly allocated on the basis of the Z-band width alone. When two independent observers classified the fibers on the basis of M-band appearance, more than 95% of the fibers were correctly classified. Thus, both the Z- and M-bands, alone or in combination, can be used as fiber type discriminators. However, the M-band structure proved to be more reliable than the Z-band width, and should therefore be used as the fiber type indicator when only one of these parameters is considered.
Immunocytochemical analysis of the rat pineal gland using antisera generated against analogs of luteinizing hormone-releasing hormone (LHRH).
Authors: Piekut D T , Knigge K M
Published: April 2011

Antisera generated against synthetic luteinizing hormone-releasing hormone (LHRH) or analogs of LHRH, agonists and antagonist, were used to stain the rat pineal gland and the median eminence of the hypothalamus. Of the LHRH analogs used, two revealed immunoreactive material in median eminence and not pineal, two stained pineal only, and three stained median eminence and pineal. Our observations suggest that the immunoreactive material in the pineal gland may be an LHRH-like substance whose affinity characteristics are different from that of hypothalamic LHRH. The affinity properties of hypothalamic LHRH for antibody appear to reside in the C-terminus of the molecule, whereas binding of the antigen in the pineal to antibody appear to be influenced by an N-terminal modification of the LHRH molecule.
A comparative study of the use of monoclonal antibodies using three different immunohistochemical methods: an evaluation of monoclonal and polyclonal antibodies against human prostatic acid phosphatase.
Authors: Naritoku W Y , Taylor C R
Published: April 2011

The use of immunohistochemical methods has been advocated for the detection and localization of prostatic acid phosphatase in paraffin sections of human prostate. This article explores the possible advantages of utilizing monoclonal antibodies in this method. Monoclonal antibodies, specific for human prostatic acid phosphatase, were integrated into three different immunohistochemical procedures. In the first method, a three-layer peroxidase-antiperoxidase (PAP) system was employed; the monoclonal antibody was followed by rabbit bridge antibody directed against mouse immunoglobulin and mouse PAP complex. The second method was a three-layer system utilizing biotin-labeled horse anti-mouse antibody as "bridge" antiserum between the primary monoclonal antibody and an avidin-biotin-horseradish peroxidase complex. The third method was a four-layer system; the monoclonal antibody was followed by rabbit anti-mouse serum, swine anti-rabbit immunoglobulin as the bridge antibody and rabbit PAP complex. It was found that some, but not all, monoclonal antibodies can be used for the detection of prostatic acid phosphatase in paraffin sections. The four-layer PAP method was found to be the most sensitive method of the three systems tested; however, the avidin-biotin method required the least amount of time. No significant difference in the quality of staining was observed between monoclonal antibodies and carefully absorbed conventional antiserum.
Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells.
Authors: Spicer S S , Sens M A , Tashian R E
Published: April 2011

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.
A fixative for use in muscle histochemistry.
Authors: Croker B P , Bossen E H , Brinn N T , Hammond F A
Published: April 2011

A fixative solution that preserves the activity of some relevant enzymes in muscle histochemistry is described. Portions of human muscle biopsy specimens and selected murine muscles were fresh frozen or placed in the fixative at room temperature for up to 1 month before freezing. Cryostat sections of fresh frozen and fixed frozen tissue were assayed for nicotinamide adenine dinucleotide phosphate (NADH)-tetrazolium reductase (NADH), several adenosine triphosphatases (ATPases), myoadenylate deaminase (MD), and phosphorylase. NADH, ATPase, and MD activity were preserved following fixation but phosphorylase was not preserved. Murine spleen and kidney were similarly tested for acid phosphatase (acid phos), alkaline phosphatase (alk phos), and nonspecific esterase (NSE). Alk phos activity was preserved but acid phos and NSE activity were significantly reduced following fixation. This fixative is useful in some circumstances for processing or shipping human muscle biopsy specimens and experimental tissues.
Histochemical identification of sulfation position in chondroitin sulfate in various cartilages.
Authors: Hasty K A , Smith G N , Kang A H
Published: April 2011

The ability of chondrocytes to synthesize chondroitin-4-sulfate (C4S) as opposed to chondroitin-6-sulfate (C6S) is a phylogenetically related phenomenon seen among adult higher vertebrates and developmentally during the embryogenesis of these vertebrates. While the embryonic cartilage may be initially a C6S matrix, C4S synthesis is seen to develop with time. We have histochemically localized these differences in sulfation with the cationic carbocyanine dye, Stains-all, in a spectrum of cartilages that vary in the sulfation position of their chondroitin sulfate. Cartilages from the rat and rabbit that are predominantly C4S stained magenta at pH 4.3, while the C6S-rich cartilage matrices from the regenerating rabbit ear and lamprey cranium stained blue. Embryonic chicken cartilages develop a gradient of magenta matrix with age, with increased concentration toward the articular surface. Both magenta and blue matrices were absent after pretreatment with chondroitinase ABC but were present after Streptomyces hyaluronidase digestion. The magenta staining was a property of the cartilage matrix as a whole, since isolated C4S and C6S stained blue. The differential staining was seen at pH 4.3, but not at pH 8.8, suggesting an interaction between the chondroitin sulfate and the adjacent tissue proteins.
Cellular localization of GM1-ganglioside with biotinylated choleragen and avidin peroxidase in primary cultured cells from rat brain.
Authors: Asou H , Brunngraber E G , Jeng I
Published: April 2011

A new technique capable of demonstrating the presence and cellular localization of the ganglioside GM1 in primary cultured cells from the brains of newborn rats is described. The method is based on the highly specific binding of biotinylated choleragen to ganglioside GM1, and takes advantage of the high affinity of avidin for biotin. Thus, the biotinylated choleragen-ganglioside GM1 complex can be visualized by the use of avidin peroxidase. The results of this nonimmunologic method indicate that the concentration of ganglioside GM1 is much lower in culture astroglial cells than in neurons and oligodendroglial cells.
Light microscopic visualization of colloidal gold on resin-embedded tissue.
Authors: Danscher G , Nörgaard J O
Published: April 2011

RNase labeled with colloidal gold was used as a model for the present technique evolved for the light microscopic localization of gold-labeled substances in semithin resin-embedded sections. Tissue sections placed on glass slides were treated with the gold-enzyme complex and subsequently exposed to a photographic developed containing silver lactate. During the development gold particles are encapsulated in growing shells of metallic silver and gradually made visible in the light microscope. The amplification method can be applied to paraffin-embedded and frozen sections as well. This technique may prove useful as a supplement to studies utilizing colloidal gold or silver as markers normally used at the electron microscopic level.
Immunohistochemistry of types I and II collagen in undecalcified skeletal tissues.
Authors: Horton W A , Dwyer C , Goering R , Dean D C
Published: April 2011

Types I and II collagen were demonstrated in semithin sections of undecalcified human endochondral growth plate, articular cartilage, and subchondral bone. The effects of several different methods for fixation, embedding, exposing of antigenic determinants, and immunoperoxidase staining were examined. Fixation in buffered formalin and paraformaldehyde-lysine-periodate solution gave more intense staining for collagens than fixation in paraformaldehyde-gluaraldehyde or Bouin's solution. Specimens embedded in Spurr epoxy resin yielded intense and uniform staining of areas known to contain the particular collagens after the resin had been removed by sodium ethoxide. The staining was enchanced following enzymatic digestion, especially with protease V (Sigma). Staining sensitivity and specificity were comparable with the indirect conjugate and double peroxidase-antiperoxidase (PAP) techniques; the PAP method was less sensitive. Embedment in methacrylate resins proved unsatisfactory because of exaggerated immunostaining of mineralized sites in comparison to unmineralized areas of the same tissues. In the growth plate specimens, type I collagen was identified in the matrices of bone, periosteum, perichondrium, and in the cytoplasm of hypertrophic and degenerative chondrocytes. Type II collagen was found uniformly throughout the cartilage matrix and in spicules of unresorbed cartilage matrix located in subchondral bone. A similar staining pattern was observed for the articular cartilage, except that type I collagen was not detected in chondrocytes.
Histochemical and ultrastructural characterization of serotonin-containing cells in rabbit tracheal epithelium.
Authors: Dey R D , Shannon W A , Hagler H K , Said S I
Published: April 2011

Tracheal endocrine cells (TECs) that contain serotonin have been characterized previously by staining with ferric ferricyanide. In the present article, the ferric ferricyanide staining reaction has been used to locate the TECs in deplasticized thick sections of Epon-embedded rabbit tracheas. Adjacent thin sections of the same cell were subsequently observed by electron microscopy. The TECs were filled with dense-core vesicles (DCVs) located in the cytoplasm between the nucleus and the lumen and also lateral to the nucleus. In a separate experiment, pieces of rabbit trachea were treated with a solution of glutaraldehyde-dichromate to demonstrate the presence of amines. High levels of chromium were detected in the DCVs by energy-dispersive X-ray analysis. The results from these studies have correlated the ultrastructure of a serotonin-containing endocrine cell present in rabbit tracheal epithelium with a cell type previously characterized only by light and fluorescence histochemical methods. The results also indicate that serotonin in these cells is stored in the DCVs.
Developmental changes in the ovarian follicular basal lamina detected by immunofluorescence and electron microscopy.
Authors: Bagavandoss P , Midgley A R , Wicha M
Published: April 2011

Alterations in the basal lamina (BL) of developing follicles were studied by immunofluorescent microscopy using antibodies against type IV collagen, laminin, and fibronectin, and by electron microscopy. Ovarian development was induced in immature rats by sequential administration of estradiol, follicle-stimulating hormone (FSH) and human chorionic gonadotropin (hCG). A continuous BL was observed in healthy follicles treated with estradiol and FSH. As determined by immunofluorescence, laminin, type IV collagen, and fibronectin were restricted to the BL and the theca but not to the granulosa. When follicles were allowed to undergo atresia or induced to ovulate with hCG, the BL became fragmented. This was confirmed by electron microscopy of healthy, atretic, and luteinizing follicles which showed that in healthy follicles the BL was continuous, whereas in both atretic and luteinizing follicles, it was fragmented. Atresia was also associated with the penetration of thecal cells into the follicles. These observations indicate that the intact BL present in healthy follicles undergo extensive changes during atresia and ovulation.
Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.
Authors: Green M R
Published: April 2011

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.
Ultrastructural differentiation within the central nervous system between indolamines and catecholamines by energy dispersive X-ray analysis following paraformaldehyde pretreatment.
Authors: McClung R E , Wood J
Published: April 2011

Dense bodies containing high amounts of chrome were localized in the perikarya of substantia nigra and dorsal raphe neurons following the cytochemical reaction of endogenous dopamine and serotonin (respectively) with glutaraldehyde-dichromate (GDC). Energy dispersive X-ray analysis of these bodies revealed chrome levels two to four times higher than those recorded from the cytoplasmic background. Pretreatment with paraformaldehyde blocked the GDC reaction within the dense bodies in the substantia nigra (chrome levels similar to background), while the chrome levels in the dense bodies of the raphe neurons remained elevated. This demonstrates that pretreatment with paraformaldehyde allows selective localization of central nervous system serotonin stores by the GDC technique.
Connective tissue elements in rat bone marrow: immunofluorescent visualization of the hematopoietic microenvironment.
Authors: Bentley S A , Foidart J M , Kleinman H K
Published: April 2011

Immunofluorescent staining of frozen sections of rat bone marrow for collagen types I and III revealed the presence of a distinctive, collagen-producing cell type. Morphologically, these cells closely resembled reticular cells. They were large, with branching cytoplasm and were closely related to an extensive intercellular matrix of collagenous material that surrounded the hematopoietic cells of the marrow. Biochemical studies demonstrated synthesis of collagen types I and III, in a ratio of 4:1, by fresh rat bone marrow cells.
Visualization of intracellular trafficking: use of biotinylated ligands in conjunction with avidin-gold colloids.
Authors: Morris R E , Saelinger C B
Published: April 2011

A simple, sensitive method to visualize the binding and internalization of protein ligands by cells in culture is described. A biotinylated toxin was used as ligand, and succinoylated avidin adsorbed onto 5.2 nm gold sols was the electron-dense marker. This method affords direct localization of proteins that are on the cell surface or intracellular without need for techniques that alter membrane integrity.
Immunocytochemical localization of Na+,K+ adenosine triphosphatase in the rat retina.
Authors: Stahl W L , Baskin D G
Published: April 2011

Immunocytochemical localization of Na+,K+-adenosine triphosphatase (ATPase) in fixed sections of rat retina was carried out using polyclonal antibodies to the highly purified enzyme and peroxidase-antiperoxidase and immunofluorescence techniques. In the photoreceptor layer of the rat retina, Na+,K+-ATPase immunoreactive sites were associated primarily with inner segments and only background activity was associated with the outer segments. This localization supports the proposed distribution of Na+,K+-ATPase in photoreceptor cells suggested by physiological studies. The Na+,K+-ATPase immunoreactive sites were also localized in the inner and outer plexiform layers, which are rich in synaptic contacts. The reticular pattern of immunostaining in those regions suggests that Na+,K+-ATPase immunoreactivity is associated with neuronal processes in those layers.
Ultrastructural localization of zinc and calcium within the granules of rat Paneth cells.
Authors: Dinsdale D
Published: April 2011

Zinc (up to 300 mmol/kg dry weight) and calcium (up to 350 mmol/kg dry weight) were demonstrated in the granules of Paneth cells from the ileum of adult rats by quantitative electron probe X-ray microanalysis of freeze-dried frozen sections. In resin-embedded samples zinc was also localized by histochemical techniques, the validity of which were subsequently evaluated by X-ray microanalysis. The concentration of zinc was highest in granules towards the apex of the cell. Large differences were found between the zinc levels of granules from rats receiving different batches of the same stock laboratory diet. These differences were eliminated by both oral and intraperitoneal administration of supplementary zinc.
Ultrastructural cytochemical identification of the siderophilic enterocyte.
Authors: Parmley R T , Barton J C , Conrad M E
Published: April 2011

Specific iron-binding sites in the gut were visualized by the sequential incubation of glutaraldehyde-fixed specimens in iron nitrilotriacetate (FeNTA) and acid ferro-cyanide (AF). In rat duodenum, jejunum, and ileum, approximately half of the enterocytes from the crypt to the distal villus contained FeNTA-AF-reactive material. The staining intensity of individual enterocytes appeared to decrease progressively in more distal locations in the gut. Maximal FeNTA-AF staining was observed in cells in the upper half of the villus, and was localized primarily in microvilli, apical cytoplasm, and lateral membranes. In duodenal crypt cells, stain deposits were present primarily in the microvilli. FeNTA-AF stained sites in the cytoplasmic and microvillus matrix were approximately 100-fold greater in number than were sites of intrinsic iron stained with AF alone. FeNTA-AF and AF staining in human duodenal enterocytes was similar to that observed in rat duodena, demonstrating the applicability of this methodology to human samples. In rat duodena, the distribution of AF staining alone in specimens taken 10 min after the in vivo intraduodenal administration of FeCl2 was similar to that of FeNTA-AF staining in tissue from fasted animals not given iron. The distribution and frequency of iron-binding sites stainable with the FeNTA-AF method which occur in a subpopulation of enterocytes can be correlated closely with physiologic, histologic, and ultrastructural parameters of inorganic iron absorption previously reported.
Multidirectional differentiation in neuroendocrine neoplasms.
Authors: DeLellis R A , Tischler A S , Wolfe H J
Published: April 2011

Histopathological and experimental observations indicate that tumors composed wholly or in part of neuroendocrine elements may arise in tissues derived from ectoderm (including neuroectoderm), mesoderm, and endoderm. These tumors frequently exhibit multidirectional differentiation as manifested by multihormonality and by the presence of morphological features indicative of divergent differentiation both in vivo and in vitro. The existence of stem cells, plasticity of differentiated cells, microenvironmental influences, and random events are factors which might all interact to determine the characteristics of any particular tumor. The production of characteristic regulatory peptide products in association with tumors of specific histological subtypes and with other neuroendocrine markers suggests mechanisms for nonrandom activation of multiple genes common to neuroendocrine-programmed cells. Future studies applying new molecular biological techniques to intact tissues and to in vitro models may help to clarify the mechanisms that regulate the expression of the neuroendocrine phenotype in normal and neoplastic states.
Development of the heterogeneous distribution of carbamoyl-phosphate synthetase (ammonia) in rat-liver parenchyma during postnatal development.
Authors: Gaasbeek Janzen J W , Moorman A F , Lamers W H , Charles R
Published: April 2011

Carbamoyl-phosphate synthetase (ammonia) is homogeneously distributed in rat-liver parenchyma at birth, as demonstrated by immunohistochemistry. A heterogeneous distribution can first be demonstrated at 6 days post partum, but can be masked by use of a too sensitive detection system. This heterogeneity is established by a decrease in enzyme content around the hepatic venules and a considerable increase in enzyme content in the remaining parenchyma. The perivenous decrease in enzyme content does not occur in all hepatocytes synchronously. The adult type of heterogeneity is characterized by a perivenous layer, only two to three cells thick, in which carbamoyl-phosphate synthetase can no longer be detected, irrespective of the sensitivity of the assay used. This situation is fully established at the age of two months.
Fine structural localization of acetylcholinesterase activity in rat submandibular gland.
Authors: Topilko A , Caillou B
Published: April 2011

Using the indirect thiocholine method, the ultrastructural localization of acetylcholinesterase (AChE) activity in the normal rat submandibular gland was studied. Cytochemical demonstration of AChE is based on coupling the hydrolysis of acetylthiocholine iodide to the precipitation of heavy metal salts. AChE-associated reaction product was selectively revealed in the perinuclear space and in the endoplasmic reticulum of the intercalated duct cells, in some cells of granular convoluted tubules, and in the striated duct epithelium, as well as in the myoepithelial cells. Although AChE activity generally occurred inside the cells, electron-dense precipitates were shown in intercellular space and in the stroma of the gland. Fine localization of AChE activity was also found in nerve bundles, predominantly between axons and between axons and Schwann cell. Our observations indicate that AChE is synthesized in the epithelium of the ducts and in the myoepithelial cells of the salivary gland. It is not known yet whether this enzyme is released from the intracytoplasmic membrane system into the extracellular space and then transported to the regions of the gland innervation. Conceivably AChE synthesized in the submandibular gland cells could also be considered an inhibitory modulator of the regulatory functions of biologically active polypeptides.
Immunoelectron microscopic localization of alpha-actinin on Lowicryl-embedded thin-sectioned tissues.
Authors: Lemanski L F , Paulson D J , Hill C S , Davis L A , Riles L C , Lim S S
Published: April 2011

A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.
Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues.
Authors: Tung P S , Domenicucci C , Wasi S , Sodek J
Published: April 2011

Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.
Measurement of DNA content in single cells morphologically identified on smears.
Authors: Maruo N , Nakabo T , Kondo M
Published: April 2011

A method was developed for measuring the nuclear DNA content in single cells previously identified on a bone marrow smear stained by the Wright-Giemsa method. The smear was first photographed and the location of individual cells, identified by morphology, was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained by the Feulgen method in 0.05% pararosaniline Schiff's reagent (pH 2.3) at 7 degrees C for 10 min. Nuclear red fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA after prior irradiation of smears with green light for 9 hr. The method is useful for measuring cell DNA content in heterogeneous cell populations when morphological cell identification is required.
Immunoelectron microscopic localization of calmodulin and phospholipase A2 in spermatozoa. I.
Authors: Weinman S , Ores-Carton C , Rainteau D , Puszkin S
Published: April 2011

Using the Lowicryl K4M embedding technique, together with indirect immunoferritin or immunogold labeling on ultra-thin sections, tubulin, calmodulin and phospholipase A2 were distinctly localized in ejaculated bull spermatozoa. Calmodulin was concentrated on the plasma membrane, nucleus, post-acrosomal substance, and, in lesser amounts, between coarse fibers and axonemal microtubules of the flagellum. Phospholipase A2 was distributed evenly along the plasma membrane, nucleus, acrosome, post-acrosomal substance, and in the flagellum, on mitochondria, fibrous sheath, coarse fibers, between coarse fibers and axonemal microtubules. Antibodies to tubulin labeled only axonemal microtubules, including the central pair of microtubules. Patterns of tubulin labeling were identical when ferritin granule- or gold particle-conjugated antibodies were tested. In agreement with our previous biochemical studies demonstrating calmodulin binding to phospholipase A2, concomitant with enhancement of phospholipase A2 activity (Arch Biochem Biophys 241:413, 1985), the overlapping distribution of calmodulin and phospholipase A2 in several parts of the sperm suggests that these proteins may play a concerted role in male gamete function in preparation for or during fertilization. The distinct distribution of tubulin along flagellum microtubules indicates their special function in sperm mobility.
Alkaline proteinase localization in myoblasts.
Authors: Stauber W T , Fritz V K , Dahlmann B , Kay J , Heath R , Mayer M
Published: April 2011

Recent interest in elucidating the role of non-lysosomal proteases in intracellular protein catabolism in muscle has led to various investigations with three alkaline proteases: a trypsin-like, a chymotrypsin-like, and a high molecular weight cysteine proteinase. Although in vitro biochemical assays have revealed the catabolic potential of at least two of these proteases, confirmation of their presence in muscle cells has been difficult. In this study immunohistochemical techniques were employed to localize each of these proteases in rat myoblasts. Antisera against the trypsin-like and chymotrypsin-like proteinase (both serine proteinases) showed strong localization in the cytoplasm immediately around the nucleus. Both also stained chromatin material in the nucleus of these cells. Fluorescent localization of the high molecular weight cysteine proteinase (Proteinase I) also appeared to be cell-associated in the myoblasts. The use of myoblasts in cell culture sections of whole muscle was advantageous, since localization of the proteases could be assessed in the absence of other cell types.
Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder.
Authors: Coleman R A , Harris H W , Wade J B
Published: April 2011

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.
Freeze-fracture cytochemistry of rat glomerular capillary tuft. Determination of wheat germ agglutinin binding sites and localization of anionic charges.
Authors: Chevalier J , Appay M D , Wang X Y , Bariety J , Pinto Da Silva P
Published: April 2011

We propose here the use of freeze-fracture to gain access and to label in vitro glomerular components and locate WGA receptors and anionic sites. Tissues are frozen, fractured under liquid nitrogen, and thawed. Freeze-fracture rendered all glomerular structures directly accessible to the reagents. This made possible study of the nature and topology of cationized ferritin and WGA binding sites. WGA-gold complexes were observed over plasma membranes of podocytes and of endothelial and mesangial cells. Labeling of podocytes and endothelial cells was similar in the mesangial area and in the peripheral part of the capillary loop. Cross-fractures of extracellular matrices showed that WGA bound uniformly to the glomerular basement membrane (GBM) as well as to mesangial matrix. In fractured specimens treated with neuraminidase, WGA was no longer observed over podocytes but it consistently labeled the surface of endothelial and mesangial cells. Whereas in GBM cross-sections WGA binding was greatly reduced or even abolished, it remained unmodified in the mesangium. This shows that only NeuNAc (sialic acid) might account for the binding of WGA to podocytes, whereas GlcNAcs appear to be the main WGA binding sites on endothelial and mesangial cells and in the mesangial matrix. Both NeuNAc and GLcNAc residues are probably associated in GBM. With cationized ferritin (pI 8.3) at pH 7.4, intense, continuous labeling was seen all over the different plasma membranes, denser in podocytes than in endothelial cells. CF was also observed in cross-fractured profiles of extracellular matrices and never appeared agglutinated in discrete sites.
An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells.
Authors: Nilsson O , Dahlström A , Geffard M , Ahlman H , Ericson L E
Published: April 2011

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.
Anti-idiotypic antibodies as a tool for cytochemical identification of substance P receptors in the central nervous system.
Authors: Conrath M , Couraud J Y , Pradelles P
Published: April 2011

Anti-idiotypic antibodies may serve as valuable probes for cytological identification of peptide receptors in the CNS. We have previously described the preparation of anti-substance P (SP) anti-idiotypic antibodies (anti-Id Ab) and have shown that they recognize SP receptors. These anti-Id Ab can be used in cytology to label SP receptors in CNS. We chose rat cervical spinal cord as a model because SP is present in large amounts in the dorsal and ventral horns, where it is implicated in pain and in motor function, respectively. After application of an indirect immunoperoxidase technique to tissue sections from perfused animals, immunolabeling was seen in the two superficial layers of the dorsal horn, the area surrounding the central canal, extending along the white matter in lamina VII, and in part of the ventral horn. This localization is in accordance with the classical distribution of SP receptors as seen by autoradiography with labeled SP. In the light of control experiments, as well as of biochemical and pharmacological arguments, we discuss the specificity of the immunolabeling. We conclude that anti-Id Ab recognize NK-P receptors, although crossreaction with NK-A or NK-B receptors cannot be totally ruled out.
Visualization of exocellular lectins in the entomopathogenic fungus Conidiobolus obscurus.
Authors: Latgé J P , Monsigny M , Prévost M C
Published: April 2011

We demonstrated the presence of lectins binding to glucose and N-acetylglucosamine on the surface of Conidiobolus obscurus spores by using glycosylated serum albumins substituted with fluorescent dyes and colloidal gold. The role of these exocellular lectins was examined in relation to the adhesion of the fungal spores to their host insect and the pathogenicity of the fungus.
In situ detection of human immunodeficiency virus (HIV) nucleic acid in H9 cells using nonradioactive DNA probes and an image cytophotometry system.
Authors: Donovan R M , Cohen S H , Peterson W R , Bolton V , Jordan G W , Carlson J R , Vanden Brink K M , Goldstein E , Bush C
Published: April 2011

Rapid and sensitive nonradioactive methods to detect human immunodeficiency virus (HIV)-infected cells are needed in clinical medicine. We developed an in situ hybridization test using 2-acetylaminofluorene (AAF)-labeled HIV DNA as a hybridization probe. Hybridized probe was detected using rabbit anti-AAF antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit, and the bromochloroindolyl phosphate-nitroblue tetrazolium reaction. An image cytophotometry system was used to quantitate the percentage of HIV-infected cells. These methods were used to determine the percentage of H9 cells infected with HIV. HIV was detected in 0% of cells on day 1 post infection, 7% on day 4, 41% on day 8, and 5% on day 15. These results paralleled those of the reverse transcriptase assay and an antigen capture ELISA assay for HIV antigen. Thus the AAF modified HIV DNA probe detected HIV nucleic acid in infected H9 cells and the image cytophotometry system improved the sensitivity and objectivity of detection.