bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical and immunocytochemical characterization of mineral binding proteoglycans in rat bone.
Authors: Takagi M , Maeno M , Kagami A , Takahashi Y , Otsuka K
Published: April 2011

We examined biochemically and immunocytochemically the type and distribution of mineral binding proteoglycans (PGs) in rat mid-shaft subperiosteal bone using three monoclonal antibodies (MAb 1-B-5, 9-A-2, and 3-B-3) which specifically recognize unsulfated chondroitin, chondroitin 4-sulfate (C4-S) and dermatan sulfate (DS), and chondroitin 6-sulfate. Bone proteins were extracted from fresh specimens with a three-step technique: 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl (E-extract), followed by GdnCl. Western blot analysis of SDS-polyacrylamide gel electrophoresis revealed that E-extract after chondroitinase ABC digestion reacted strongly with MAb 9-A-2 but not with MAb 1-B-5 or 3-B-3. After adehyde fixation, ethanolic trimethylammonium EDTA was used as a demineralizing agent for light and electron immunocytochemistry. This provided good retention of water-soluble PGs in the specimens. After chondroitinase ABC pre-treatment of tissue sections, MAb 9-A-2 specifically stained C4-S and/or DS in the walls of osteocyte lacunae and bone canaliculi in the mineralized matrix as well as in the unmineralized matrix such as pre-bone, vascular canals, and pericellular matrix surrounding osteocytes; the remainder of the mineralized matrix lacked staining. These results indicate that mineral binding PGs contain C4-S and/or DS and are exclusively localized in the walls of the bone lacuna and canaliculus.
Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT.
Authors: Kooij A , Frederiks W M , Gossrau R , Van Noorden C J
Published: April 2011

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.
Endocytosis in cultured rat alveolar type II cells: effect of lysosomotropic weak bases on the processes.
Authors: Kalina M , Socher R
Published: April 2011

We investigated the uptake of Lucifer yellow and surfactant complexed with gold (S-G) by isolated alveolar Type II cells. The fluid phase marker Lucifer yellow did not reach lamellar bodies (LB) even after prolonged incubation time, whereas S-G was internalized and found in LB. Treatment of Type II cells with lysosomotropic weak bases (NH4Cl and chloroquine) resulted in dilation of endosomes, lysosomes, and LB. The effect of these agents on LB resulted in disappearance of their lamellar organization, as detected by polarized light and electron microscopy. After incubation in lysosomotropic agent-free medium, endocytosis of Lucifer yellow and S-G in treated cells was mainly directed towards large vacuoles resembling either multivesicular bodies (MVB) or lysosomes. The possible relationship between LB, MVB, and lysosomes in freshly isolated as well as cultured alveolar Type II cells is discussed.
A specific ultrastructural method to reveal DNA: the NAMA-Ur.
Authors: Testillano P S , Sanchez-Pina M A , Olmedilla A , Ollacarizqueta M A , Tandler C J , Risueño M C
Published: April 2011

We have developed a new, simple, and reproducible cytochemical method to specifically stain DNA at the electron microscopic level: the NAMA-Ur. It is based on the extraction of RNA and phosphate groups from phosphoproteins by a weak alkali hydrolysis (NA) which does not affect DNA, followed by blockage of the amino and carboxyl groups by methylation and acetylation (MA). Finally, sections are stained by uranyl (Ur), which can bind only to DNA. The efficiency of the pre-treatment (NA and MA) was demonstrated by X-ray microanalysis at the transmission electron microscopic level. The NAMA-Ur method has been successfully performed en bloc and on Lowicryl sections in mammalian and plant cells. A specific contrast is observed in the DNA-containing structures after this method, whose sensitivity allows visualization of electron-dense chromatin fibers of 10-12 nm composed of 3-nm DNA fibrils. This staining method has been combined with anti-DNA antibodies, providing complementary information to detect DNA in situ. We propose the NAMA-Ur as an easy method to investigate the chromatin organization in situ at the ultrastructural level.
Surfactant protein A is localized at the corners of the pulmonary tubular myelin lattice.
Authors: Voorhout W F , Veenendaal T , Haagsman H P , Verkleij A J , van Golde L M , Geuze H J
Published: April 2011

Immunogold labeling on sections of a freeze-substituted tubular myelin-enriched fraction isolated from a bronchoalveolar lavage of rat lung showed that surfactant protein A (SP-A) occurs predominantly at the corners of the tubular myelin lattice. Seventy-nine percent of the gold particles were located within 20 nm from a corner. Extracellular SP-A was detected only in the tubular myelin lattice and not in vesicles or secreted lamellar bodies. Ultra-thin cryosections of rat lung fixed in vivo showed that intracellular SP-A was distributed homogeneously over the stacked membranes of lamellar bodies in alveolar Type II cells. The presence of SP-A at the corners of the tubular myelin lattice suggests an important role of this protein in the formation and/or maintenance of this highly ordered lattice.
Immunoelectron microscopic evidence for organ differences in the composition of peroxisome-specific membrane polypeptides among three rat organs: liver, kidney, and small intestine.
Authors: Usuda N , Kuwabara T , Ichikawa R , Hashimoto T , Nagata T
Published: April 2011

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.
Binding and endocytosis of thrombospondin and thrombospondin fragments in endothelial cell cultures analyzed by cuprolinic blue staining, colloidal gold labeling, and silver enhancement techniques.
Authors: Völker W , Schön P , Vischer P
Published: April 2011

We investigated the distribution of thrombospondin-specific binding sites and the uptake of thrombospondin-gold conjugates in cultured porcine endothelial cells by light and electron microscopy. Colloidal gold marker and silver enhancement techniques were applied for cytochemical detection of monomeric thrombospondin and fragments of thrombospondin. Thrombospondin binds to granular and fibrillar structures and to sites of cell-cell contact on the cell surface, as indicated by many proteoglycan-cuprolinic blue precipitates. Cell migration tracks on the culture dish bottom are most heavily stained. Labeling of intact thrombospondin and of proteolytic fragments of thrombospondin with colloidal gold followed by silver intensification enables one to detect its binding and uptake in endothelial cells. Binding to the cell surface and uptake of thrombospondin-gold particles was inhibited by heparin but not by hyaluronic acid or chondroitin sulfate. The heparin binding region at the N-terminal end of the thrombospondin molecule proved to be essential for cell surface binding. Gold-conjugated thrombospondin fragments devoid of the heparin binding region were not internalized. After 60 min incubation at 37 degrees C, thrombospondin-gold particles accumulated in the lysosomal compartment close to the nucleus. In the presence of monensin and ammonium chloride, vesicles in this area are swollen and the concentration of particulate marker is reduced. Binding and uptake of thrombospondin by vascular endothelial cells appears to require linkage of the heparin binding region of the thrombospondin molecule to coated pits and heparan sulfate-rich molecules as receptors. Colloidal gold conjugation of thrombospondin fragments proved to be useful for cytochemical characterization of molecular domains.
Immunogold electron microscopy of soluble proteins: localization of Bet v I major allergen in ultra-thin sections of birch pollen after anhydrous fixation techniques.
Authors: Grote M
Published: April 2011

To localize the highly water-soluble major allergen Bet v I in ultra-thin sections of birch pollen, pollen grains were cracked, air-dried, and processed for electron microscopy using one of the following preparation techniques: fixation in aqueous p-formaldehyde + cetylpyridinium chloride; fixation in p-formaldehyde vapor; fixation in benzoquinone vapor; inert dehydration; or no fixation. Afterwards the pollen grains were embedded in Lowicryl K4M resin at low temperature. Ultra-thin sections were cut and incubated with a monoclonal antibody against Bet v I, followed by a gold-labeled secondary antibody. In some experiments, commercial rabbit IgG antibodies against birth pollen allergens were also used, followed by incubation with the protein A-gold complex. Bet v I could be localized only after vapor fixation and in the inert dehydrated specimens. Best preservation of ultrastructure and antigenicity was obtained after p-formaldehyde vapor fixation. Bet v I antibody binding sites were detected only in the cytoplasmic matrix of the pollen grain, never in the pollen wall. Commercial rabbit antibodies bound to cytoplasm and wall of all prepared specimens, even after aqueous fixation. This might be explained by the assumption that these antibodies recognize a variety of antigenic and allergenic structures, not all of which are so highly soluble as Bet v I.
Acid phosphatase activity is detected preferentially in the osteoclastic lineage by pre-treatment with cyanuric chloride.
Authors: Nakamura Y , Yamaguchi A , Ikeda T , Yoshiki S
Published: April 2011

We previously reported a simple method to detect osteoid matrices in decalcified bone sections by pre-treatment with cyanuric chloride. We have applied this technique to identify osteoclasts and their precursors in rats. In JB-4 sections prepared from untreated bone tissues with cyanuric chloride, both acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) were found not only in osteoclasts and bone marrow mononuclear cells but also in osteoblasts. In contrast, treatment of bones with cyanuric chloride resulted in staining ACP preferentially in osteoclasts and mononuclear cells adjacent to the bone surface. In the osteoclasts and most of the ACP-positive mononuclear cells, autoradiography showed calcitonin binding. Decalcification with EDTA did not affect the staining for ACP activity in bones treated with cyanuric chloride. It was possible to simultaneously identify ACP and osteoid matrix in a decalcified section. In soft tissues without treatment with cyanuric chloride, both ACP and TRAP were detected in splenic macrophages, alveolar macrophages, and proximal convoluted ducts in kidney. Neither ACP nor TRAP was found in these cell types in the tissues treated with cyanuric chloride. This procedure provides a new, simple method to identify a more restricted population in the osteoclastic lineage than that detected by TRAP staining.
In vitro bromodeoxyuridine labeling of nuclei: application to myotube hybridization.
Authors: Labrecque C , Huard J , Dansereau G , Albert N , Tremblay J P
Published: April 2011

Rat myoblast nuclei were labeled with various concentrations of bromodeoxyuridine (BrdU), an analogue of thymidine, for 24 or 48 hr. Almost every myoblast was labeled with BrdU at concentrations between 10(-7) M and 10(-5) M. When the cells were labeled with 0.5 microM or more, the percentage of labeled cells remained over 90% and 80% at 2 and 5 days, respectively. However, when the cells were labeled with BrdU concentration lower than 10(-7) M the percentage of labeled nuclei decreased more rapidly with time. The BrdU-labeled cells were mixed with an unlabeled population to determine whether their capacity to fuse was reduced. At a BrdU concentration of 0.5 x 10(-6) M, labeled myoblasts fused to a similar extent as unlabeled myoblasts, and a high percentage of marked cells were still perceptively labeled after 5 days. In contrast, the fusion capacity of myoblasts incubated with more than 10(-6) M BrdU was inhibited after only few rounds of DNA synthesis. These myoblasts were eventually able to fuse, however, when the BrdU diminished in the DNA due to cell division. These results indicate that labeling with BrdU at a concentration of 0.5 x 10(-6) M and an incorporation time of 48 hr is optimal to obtain perceptible immunocytochemical staining without affecting myoblast fusion. Such BrdU immunolabeling could be used as a nuclear marker for hybridization studies.
Na,K-ATPase expression in the developing brine shrimp Artemia. Immunochemical localization of the alpha- and beta-subunits.
Authors: Sun D Y , Guo J Z , Hartmann H A , Uno H , Hokin L E
Published: April 2011

Developing brine shrimp are a good experimental model for study of gene expression during development. Development is initiated on suspension of brine shrimp cysts in seawater. Only 48 hr are required for progression from cyst to the larval stage. We have localize the alpha- and beta-subunits in different cells by immunostaining as development progresses. Both alpha- and beta-subunits are first detected in epidermal cells in the trunk region at the emergence 2 stage (16-hr incubation). At the nauplius 1 stage (24 hr) the enzyme appears in the brain and epidermal regions, as well as in mesenchymal cells, with weaker staining in the salt gland. After further development (nauplius 2 stage, 36 hr) stronger staining appears in the salt gland and in the epidermal region. At the nauplius 3 stage (48 hr) the enzyme appears in the midgut mucosa. Co-localization of the alpha- and beta-subunits appears in all positive cells during development. In the epidermal and salt gland cells the enzyme is mainly localized on the basolateral membrane. The basolateral localization of the Na,K-ATPase in epidermal and salt gland cells suggests that Na+ is actively transported into the epidermal and salt gland cells and passively diffuses out from the apical region.
Ultracytochemical characteristics of cultured goat brain microvascular endothelial cells [corrected]
Authors: Vorbrodt A W , Trowbridge R S
Published: April 2011

This ultrastructural study was undertaken to determine the localization of cytochemically demonstrable blood-brain barrier (BBB)-associated enzymatic activities and of some nonenzymatic constituents in goat [corrected] brain microvascular endothelial cells (ECs) growing in vitro. Positive reactions for alkaline phosphatase (AP), 5'-nucleotidase (5'N), transport ATPase (Na+,K(+)-ATPase), and adenosine diphosphatase (ADPase) were present on both apical and basolateral plasma membranes (PMs) of the ECs. The reaction for calcium-dependent ATPase (Ca(2+)-ATPase) was less intense and was restricted to basolateral PM and associated plasmalemmal pits. These cells also revealed an abundance of anionic sites labeled with cationic colloidal gold (CCG) and Ricinus communis agglutinin 120 (RCA)-binding sites, specific for beta-D-galactosyl residues, on the apical PM. The labeling of the apical PM with Ulex europaeus agglutinin (UEA)-gold complex, specific for alpha-L-fucosyl residues, was negligible. When compared with results of cytochemical examination of the ECs of goat [corrected] brain capillary in vivo, these observations indicate that although cells cultivated in vitro retain at confluence the enzymatic activities typical for BBB-type ECS, they lose their characteristic (polar) localization. This loss is interpreted as a reflection of lost functional polarity of the microvascular endothelium in vitro resulting from deprivation of the normal influence of the components of brain parenchyma.
Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.
Authors: Fujimoto T , Lee K , Miwa S , Ogawa K
Published: April 2011

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.
Neuronal protein NP185 in avian and murine cerebellum: expression during development and evidence for its presence in nerve endings.
Authors: Perry D G , Hanson V , Benuck M L , Puszkin S
Published: April 2011

The neuronal protein NP185 is a neural tissue-specific protein isolated from clathrin-coated vesicles in brain. Using 8G8, a monoclonal antibody (MAb) characterized in our laboratory, we studied the expression and distribution of neuronal protein NP185 in developing avian cerebellum and in mature murine cerebellum. Furthermore, we compared these parameters to that of synapse-specific neuronal protein, synaptophysin, and an axon-specific (i.e., non-synaptic) neuronal protein, neurofilament NF68. We found that NP185 expression temporally and spatially corresponds to avian cerebellar synaptogenesis. In addition, NP185 distribution parallels synaptophysin distribution throughout development, while differing from that of either unassembled or filamentous forms of NF68. The evidence also suggests that embryonic NP185 expression coincides with synaptogenesis, and that NP185 remains concentrated in the terminal boutons of mature neurons. The synapse specificity of NP185 and the recent biochemical properties reported for this protein support the postulate that this molecule may trigger synaptic events and distinguish structurally and functionally active synapses.
Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: a cytochemical, immunocytochemical, and in situ hybridization approach.
Authors: Motte P M , Loppes R , Menager M , Deltour R
Published: April 2011

We report the 3-D arrangement of DNA within the nucleolar subcomponents from two evolutionary distant higher plants, Zea mays and Sinapis alba. These species are particularly convenient to study the spatial organization of plant intranucleolar DNA, since their nucleoli have been previously reconstructed in 3-D from serial ultra-thin sections. We used the osmium ammine-B complex (a specific DNA stain) on thick sections of Lowicryl-embedded root fragments. Immunocytochemical techniques using anti-DNA antibodies and rDNA/rDNA in situ hybridization were also applied on ultra-thin sections. We showed on tilted images that the OA-B stains DNA throughout the whole thickness of the section. In addition, very low quantities of cytoplasmic DNA were stained by this complex, which is now the best DNA stain used in electron microscopy. Within the nucleoli the DNA was localized in the fibrillar centers, where large clumps of dense chromatin were also visible. In the two plant species intranucleolar chromatin forms a complex network with strands partially linked to chromosomal nucleolar-organizing regions identified by in situ hybridization. This study describes for the first time the spatial arrangement of the intranucleolar chromatin in nucleoli of higher plants using high-resolution techniques.
Antiserum to lucifer yellow: preparation, characterization, and use for immunocytochemical localization of dye-filled retinal neurons.
Authors: Brandon C , Criswell M H
Published: April 2011

We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.
Synthetic guinea pig insulin B-chain C-terminal decapeptide: a novel immunogen for generating immunocytochemical-grade antisera.
Authors: Maimon J , Mauro M , Gorray K C , Schneider B S
Published: April 2011

Antisera to guinea pig insulin are not commonly available, largely because of the short supply and limited immunogenicity of the intact hormone. To overcome these problems we have employed a novel reagent, synthetic guinea pig insulin B-chain C-terminal decapeptide, as a hapten for raising antibodies that react with intact guinea pig insulin. The decapeptide, coupled to bovine serum albumin, was successfully used as an immunogen in rabbits. The resulting anti-serum was employed for immunocytochemical staining of guinea pig insulin in pancreatic sections. The specificity of the staining was verified by both pre-absorption and pre-immune serum controls. The utility of this new antiserum for investigations of guinea pig insulin physiology is discussed.
Gold enhancement of gold-labeled probes: gold-intensified staining technique (GIST).
Authors: Morris R E , Ciraolo G M , Saelinger C B
Published: April 2011

In this report we present a staining method in which gold chloride is used to enhance the size of gold colloids. We show the utility of this technique when used in conjunction with small gold colloids, i.e., 5 nm, 4 nm, and 2.6 nm. Post-embedding staining of epoxy-embedded, gold-labeled mouse LM fibroblasts showed that staining with 0.1% gold chloride facilitated the visualization of the smallest gold colloids.
Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase.
Authors: Szabolcs M J , Windisch A , Koller R , Pensch M
Published: April 2011

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.
Ca(2+)-ATPase and Mg(2+)-ATPase in Aplysia glial and interstitial cells: an EM cytochemical study.
Authors: Maggio K , Watrin A , Keicher E , Nicaise G , Hernandez-Nicaise M L
Published: April 2011

The localization of Ca(2+)- and Mg(2+)-ATPases was determined in Aplysia central and peripheral nervous system, using an electron microscopic cytochemical method. The enzyme activity appeared localized to the membrane of glial granules (gliagrana), particularly in the peripheral nervous system of the esophagus, and on the plasma membrane of central glial cells adjacent to neuronal cell bodies. No calcium- and/or magnesium-ATPase activity was detectable on the plasma membrane of glial cells surrounding nerve axons in the pleuro-visceral connectives. These findings are discussed along two main lines: (a) the calcium-ATPase of the gliagrana coincides with a high intragranular calcium and/or proton concentration; and (b) the presence of a calcium-ATPase activity at the glio-neuronal interface around the neuronal cell bodies coincides with the use of calcium ions as charge carriers of the action potential, and its absence at the level of the axon with the concurrent functional use of sodium ions.
Use of the dinitrophenyl hapten sandwich staining procedure (DHSS) to localize estrogen receptors in paraffin-embedded tissues.
Authors: Gee J M , Amselgruber W M , Jasani B , Nicholson R I
Published: April 2011

To date, reliable and sensitive methods to localize the estrogen receptor (ER) in rat tissues and human breast cancers have required the use of frozen sections. This not only incurs poor tissue structure but also precludes the study of small breast lesions that are usually paraffin embedded for histological evaluation. We have developed and optimized a dinitrophenyl hapten sandwich staining (DHSS) immunocytochemical procedure to demonstrate ER in paraffin-embedded, hormone-sensitive tissues of the rat and in human breast cancers. The method was applicable to formalin- and Bouins-fixed material, with trypsinization of sections being essential. The immunocytochemical system utilized a dinitrophenyl (DNP) hapten-labeled monoclonal antibody to the receptor. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase complex, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This highly sensitive method localized the ER within paraffin-embedded rat uterus, fallopian tube, vagina, and normal and cancerous mammary gland. Furthermore, excellent staining was generated in human breast cancers in accordance with their ER-ICA status. Control sections involving simultaneous incubation with DNP-labeled and unlabeled H222 were background free, while uteri from castrated rats demonstrated reduced receptor immunostaining. Staining was also absent in ER-negative human breast tumors.
Localization and synthesis of alpha-fetoprotein in chorioallantoic membrane from chick embryo.
Authors: Sanchez Palazon L , Rodriguez-Burgos A
Published: April 2011

Alpha-fetoprotein (AFP) is a major globulin of the embryonic serum of mammals, birds, and other vertebrates. It is synthesized chiefly by the liver and/or the yolk sac. The aim of this work was to confirm the occurrence of AFP in the chorioallantoic membrane (CAM) from 14-day chick embryo. AFP had previously been detected by immunoelectrophoresis in CAM extracts under the suspicion that it could be a mere artifact resulting from blood contamination. The immunohistochemical study of the CAM carried out for this purpose revealed the protein to be solely located in the mesodermal layer. The joint use of organ culture and immunoperoxidase techniques has enabled us to find evidence for the synthesis of AFP in the cells of this layer. These results confirm the occurrence of such a significant carrier globulin to embryonic development in one more tissue that can be added to the short list of AFP-producing tissues.
Rumenectomy-induced proliferation in duodenal villous epithelium is mechanistically related to the disappearance of statin, a non-proliferation-specific nuclear protein.
Authors: Kyzer S , Mitmaker B , Gordon P H , Schipper H M , Wang E
Published: April 2011

We undertook this study to determine the effect of rumenectomy (a known cause of duodenal crypt cell hyperplasia) on the epithelial growth kinetics of the crypt-villus axis in rat duodenum. Ten rats were randomly assigned to control (gastrotomy) and experimental (rumenectomy) groups. After 14 days rats were sacrificed and representative sections were stained with the monoclonal antibody to statin, a non-proliferation-specific protein, by the immunoperoxidase procedure. In the control group, the mean percentages of statin-positive cells in the proximal duodenum, distal duodenum, proximal jejunum, and distal jejunum were 79 +/- 8.5, 79.5 +/- 5.7, 85 +/- 1.4, and 83.5 +/- 0.7, respectively. In the rumenectomy group, statin-positive nuclei were found in the region of the villous apices only, and the corresponding values for the above four areas were 26.2 +/- 4.9, 24.5 +/- 3.5, 31.7 +/- 4.5, and 80.5 +/- 2.1. Except for distal jejunum, the differences in statin expression in the control and experimental groups were significant (p less than 0.001). Rumenectomy leads to the disappearance of statin from the villous column cells of the duodenum and proximal small bowel. The lack of expression of statin in the rumenectomy group documents the potential usefulness of this measure in future studies in neoplasia were understanding of the proliferative status is of crucial importance.
Expression of transforming growth factor beta 1 in the submandibular gland of the rat.
Authors: Amano O , Tsuji T , Nakamura T , Iseki S
Published: April 2011

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.
Histochemical quantitation of gamma-glutamyl transferase in human leukemia cell lines.
Authors: Kobb S M , Miller A M
Published: April 2011

The enzyme gamma-glutamyl transferase (gamma-GT) is involved in many biochemical systems, including the signal transduction of hematopoietic growth factors. Standard colorimetric gamma-GT assays require larger cell numbers than may be obtainable in many cases, such as with highly purified stem-cell populations. To study gamma-GT expression in limited populations, we used a histochemical stain to analyze gamma-GT semiquantitatively in cells of hematopoietic origin. Several human leukemic cell lines, including one with inducible increases in gamma-GT, were stained for gamma-GT and graded 0 through 4+ for the amount of positive granules. The gamma-GT activity demonstrated by this stain was found to be directly proportional to the gamma-GT activity obtained with a colorimetric assay and could be used to calculate approximate gamma-GT activity. This stain therefore provides a useful method for determining gamma-GT activity when limited cell numbers are available.
Ultrastructural localization of HTLV-I gag proteins p19 and p24 by single and double immunogold labeling.
Authors: Stransky G , Gay S
Published: April 2011

We developed a post-embedding immunogold labeling procedure for the ultrastructural localization of the HTLV-I gag proteins p19 and p24 by the use of monoclonal antibodies (MAb). Both antigens were shown to withstand fixation with 1% glutaraldehyde. In addition, p19 antigenicity was found not to be affected by post-fixation with 1% osmium tetroxide. The choice of resin played a decisive role in the retention of antigenicity. P19 was preserved in Lowicryl K4M as well as in LR White, whereas p24 was preserved only in Lowicryl. Both p19 and p24 were found to be localized on the HTLV-I virions themselves, whereas no positive immunostaining could be observed on the infected cells. In Lowicryl-embedded samples, in which both antigens had been preserved, a double immunogold labeling procedure was performed that allowed the co-localization of p19 and p24 on the same section. In osmicated LR White-embedded samples the quality of ultrastructural preservation of HTLV-I virions was found to be comparable to results obtained with the traditional glutaraldehyde-osmium tetroxide-epoxy resin processing.
Identification of phospholipids in secretory granules of human submucosal gland respiratory cells.
Authors: Girod S , Fuchey C , Galabert C , Lebonvallet S , Bonnet N , Ploton D , Puchelle E
Published: April 2011

Although it has been shown that tracheal epithelial cells in culture synthesize and secrete phospholipids, no direct evidence for in situ phospholipid storage in human respiratory secretory epithelial cells has been demonstrated. We used a high-resolution cytochemical enzyme-gold technique to identify and precisely localize phospholipids in human submucosal gland secretory cells. In addition, lysozyme, a specific serous cell marker, was identified using the biotinstreptavidin gold technique with lysozyme antiserum. This double labeling of phospholipids and lysozyme was performed using gold particles of diameters 15 nm and 5 nm, respectively. Quantitation of phospholipid labeling was performed on an image analyzer. Phospholipids were identified in serous granules (8.87 +/- 2.21 gold particles/microns 2) in a significantly (p less than 0.05) higher density than in mucous granules (5.57 +/- 3.07 gold particles/microns 2). These results support the hypothesis that submucosal human airway serous and mucous secretory cells produce phospholipids which may be secreted in the airway lumen.
Chromogranins or chromogranin-like proteins are present in lamellar bodies and pulmonary surfactant of rat alveolar type II cells.
Authors: Kalina M , Grimelius L
Published: April 2011

Rat alveolar Type II cells were immunostained with antibodies directed against chromogranin A (monoclonal, LK2H10) and chromogranins A and B (polyclonal, LKZM1U). The chromogranins or chromogranin-like proteins were identified in cells in lung tissue sections and isolated Type II cells at the light and electron microscopic levels. We used post-embedding immunoelectron microscopy, with immunogold, to detect the proteins' immunoreactivity in osmicated tissues. Gold particles were distributed over the phospholipid lamellae within the lamellar bodies of alveolar Type II cells and over the lattice structure of tubular myelin. Quantitative analysis of gold labeling densities in the various cell compartments indicated that only the latter two structures were specifically labeled. Controls, which included pre-absorption of both anti-chromogranin antibodies with excess chromogranin A or with native surfactant, resulted in a greater than 60% decrease in gold labeling. A possible role of chromogranins or chromogranin-like proteins as Ca2+ binding proteins in alveolar Type II cells is discussed.
Effects of formaldehyde fixation on protein secondary structure: a calorimetric and infrared spectroscopic investigation.
Authors: Mason J T , O'Leary T J
Published: April 2011

We investigated the effects of formaldehyde fixation on the secondary structure of isolated proteins (bovine serum albumin, ribonuclease A, and hemoglobin) using high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Whereas thermograms obtained by scanning calorimetry on unfixed purified proteins demonstrated denaturation transitions in the 70-90 degrees C temperature range, the thermograms showed no denaturation transitions in this temperature range when the proteins had been placed in formaldehyde solutions. Thus, fixation destroyed the denaturation transition of bovine serum albumin, ribonuclease A, and hemoglobin. Infrared spectra obtained on the unfixed and fixed proteins were essentially identical. This demonstrates that the "fixed" proteins retain the secondary structure present before fixation. We therefore conclude that the cross-linking of proteins that occurs in the process of formaldehyde fixation "locks in" the secondary structure of these protein molecules.
Increased sensitivity of the indirect immunofluorescence method by use of a tertiary fluorochrome-labeled antibody.
Authors: Svensson M A
Published: April 2011

The standard protocol for indirect immunofluorescence was modified by the addition of a third layer of antibodies. These antibodies were conjugated with the same fluorochrome as the secondary antibodies. This modification resulted in significant enhancement of the sensitivity of immunolabeling. The method may be useful when the standard indirect immunofluorescent technique results in weak labeling, and also to regain labeling in sections where specific fluorescence has faded.
Monoclonal antibodies against bovine type IX collagen (LMW fragment): production, characterization, and use for immunohistochemical localization studies.
Authors: Ye X J , Terato K , Nakatani H , Cremer M A , Yoo T J
Published: April 2011

Four high-affinity monoclonal antibodies (MAb) which react specifically with the low molecular weight (LMW) fragment of bovine type IX collagen (BIX) have been produced in mice. On the basis of the ability of these MAb to cross-react with type IX collagen purified from human, rat, and chick cartilage and to inhibit one another in a competitive inhibition assay, we conclude that the MAb D1-9, B3-1, and B2-7 recognize unique epitopes, whereas MAb B4-5 recognizes the same epitope as B3-1. None of the MAb reacted with bovine type I, II, and XI collagen. MAb D1-9 and B3-1 were tested for their ability to bind to tissue antigen, using an immunohistochemical assay system. Positive immunoperoxidase reactions were observed in the perichondrocytic regions of human and rat costochondral cartilage. Positive responses were also detected in rat auricular cartilage, as well as in tissue obtained from the middle and inner ears of rats and mice. This report demonstrates the relative ease of producing MAb to heterologous type IX collagen and the utility of these MAb for localizing type IX collagen in cartilage and cartilage-like tissues.
Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.
Authors: Ben-Ezra J , Johnson D A , Rossi J , Cook N , Wu A
Published: April 2011

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.
Monoclonal antibodies recognize localized antigens in the eye and central nervous system of the marine snail Bulla gouldiana.
Authors: Bedian V , Chen Y L , Roberts M H
Published: April 2011

The eyes of the marine snail Bulla gouldiana act as circadian pacemakers. The eyes exhibit a circadian variation in spontaneous optic nerve compound action potential frequency in constant darkness, and are involved in controlling circadian rhythms in behavioral activity expressed by the animal. To initiate an investigation of the molecular aspects of circadian rhythmicity in the Bulla eye and to identify specific molecular markers in the nervous system, we raised monoclonal antibodies (MAb) to the eye and screened them for specific patterns of staining in the eye and brain. Several MAb recognize antigens specific to groups of neurons in the brain, whereas others stain antigens found only in the eye. In addition, some antigens are shared by the eye and the brain. The antigens described here include molecules that mark the lens, retina, neural pathways between the eye and the brain, specific groups of neurons within the central ganglia, and an antigen that is shared by basal retinal neurons (putative ocular circadian pacemaker cells) and glia. These molecular markers may have utility in identifying functionally related groups of neurons, elucidating molecular specializations of the retina, and highlighting pathways used in transmission of information between the retina and the brain.
Age-dependent reversal of the lobular distribution of androgen-inducible alpha 2u globulin and androgen-repressible SMP-2 in rat liver.
Authors: Mancini M A , Chatterjee B , Roy A K
Published: April 2011

Hepatocytes situated at pericentral and periportal zones of the liver lobule show differences in the expression of several liver-specific genes, such as androgen-inducible alpha 2u globulin and androgen-repressible senescence marker protein-2 (SMP-2). A marked temporal difference in the expression of these two androgen-regulated genes has also been observed. The liver of the pre-pubertal male rat is insensitive to androgen, and during this period hepatocytes synthesize only SMP-2. During young adult life (greater than 40 days), the liver becomes androgen sensitive and concomitant synthesis of alpha 2u globulin and repression of SMP-2 occur. In the senescent male rat (greater than 750 days), the liver again becomes androgen insensitive when the decline in alpha 2u globulin is accompanied by an increase in SMP-2 synthesis. In this article we present results to show a correlation between the temporal and spatial (intralobular) changes in the expression of the androgen-inducible alpha 2u globulin and the androgen-repressible SMP-2 in rat hepatocytes. Results indicate that the temporal changes in hepatic androgen sensitivity are dictated by the intralobular location of the hepatocytes. Hepatocytes located around the central vein (pericentral/perivenous) may benefit from a paracrine advantage for the expression of a subset of genes, including the gene for the androgen receptor.
Distribution of glycoconjugates in ion transport cells of gerbil inner ear.
Authors: Sugiyama S , Spicer S S , Munyer P D , Schulte B A
Published: April 2011

Ion transport cells in gerbil inner ear were differentiated histochemically by staining glycoconjugates (GCs) with a battery of horseradish peroxidase-conjugated lectins. Strong staining with PSA and LCA showed a high content of N-linked oligosaccharides in transport cell GCs. Reactivity with PHA-L and PHA-E identified GC with triantennary and with bisected biantennary N-linked oligosaccharides, respectively, in these cells. High affinity for DSA and PWM demonstrated abundant N-acetyl lactosamine in N-linked side chains. Ion transporting epithelial cells reacting with lectins specific for N-linked oligosaccharides included strial marginal cells and outer sulcus cells of the cochlea and dark cells, transitional cells, and planum semilunatum cells of the vestibular system. In general, all of the inner ear transport epithelial cells revealed a similar lectin binding profile, with the one exception that SBA reacted strongly with ion transporting cells in the vestibular system but only weakly with those in the cochlea. Fibrocytes specialized for ion transport located in distinct areas in the suprastrial and inferior regions of the spiral ligament also stained with lectins that demonstrate N-glycosylation. However, transport fibrocytes differed from transport epithelial cells in two ways. First, they reacted e with HPA, DBA, VVA, and SJA specific for O-linkages and second, they failed to react with UEA I. The staining pattern for N-glycosylated GC resembled that for Na+, K(+)-ATPase in inner ear, suggesting a relationship between these constituents.