bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunodetection of RNA on ultra-thin sections incubated with polyadenylate nucleotidyl transferase.
Authors: Thiry M
Published: April 2011

A new method is described for locating RNA on ultra-thin sections. Sections of aldehyde-fixed, plastic-embedded cells were incubated in a medium containing polyadenylate nucleotidyl transferase (PnT) and biotinylated ATP. The labeled nucleotides bound to RNA at the surface of the ultra-thin sections were than visualized by an indirect immunogold labeling technique. The resulting labeling pattern was dependent on the presence of divalent cations in the PnT medium. The method revealed with great precision the specific RNA-containing structures within Ehrlich tumor cells. The method is applicable to Epon sections. However, the labeling intensity varies according to the fixation used. Best results were obtained on acetylated cell sections. The method can be combined with EDTA regressive staining. The in situ PnT method provides a very useful tool for pinpointing the precise location of RNA within biological material at the ultrastructural level.
Photoconversion and electron microscopic localization of the fluorescent axon tracer fluoro-ruby (rhodamine-dextran-amine).
Authors: Schmued L C , Snavely L F
Published: April 2011

Fluoro-Ruby, the fluorescent tetramethylrhodamine-dextran-amine used to demonstrate anterograde axon transport, has been successfully photoconverted and subsequently localized by electron microscopy. The photoconversion was accomplished by irradiating the tissue with green light while bathing it in a solution containing DAB. The tissue could then be examined by brightfield microscopy or processed for conventional electron microscopy. Potential advantages of the technique include greater permanence and contrast at the light microscopic level and the ability to resolve synaptic connectivity at the electron microscopic level.
Cytochemical evidence for the presence of phospholipids in epithelial tight junction strands.
Authors: Kan F W
Published: April 2011

Previous freeze-fracture experiments using either glutaraldehyde-fixed and cryoprotected specimens or unfixed rapid-frozen samples led to the proposal that cylindrical strands of the tight junction (TJ) observed in freeze-fracture preparations are inverted cylindrical micelles made up of membrane lipids and, possibly, membrane proteins. However, no one has yet been able to directly label the structural fibrils of the TJ. To test the hypothesis that TJ strands observed on freeze-fracture preparations are composed at least partially of lipids, we have combined the phospholipase A2-gold and the fracture-label techniques for localization of phospholipids. Phospholipase A2, purified from bee venom, was adsorbed on gold particles and used for specific labeling of its substrate. Phospholipase A2-colloidal gold (PLA2-CG) complex was applied to freeze-fractured preparations of rat exocrine pancreatic cells and testicular Sertoli cells, both of which are known to have extensive TJ complexes on their plasma membranes. Fracture-label replicas of exocrine pancreatic cells revealed specific association of gold particles with TJ fibrils on the protoplasmic fracture-face of the plasma membrane. The majority of these gold particles were observed either directly on the top of the TJ fibrils or adjacent to these cylindrical structures. A high density of PLA2-CG labeling was also observed over the complementary exoplasmic fracture-face of the TJ complex. This intimate association of PLA2-CG labeling with the TJ is particularly evident in the Sertoli cell plasma membrane, where rows of gold particles were observed to be superimposed on parallel arrays of cylindrical strands of the TJ complex. The present findings provide direct cytochemical evidence to support the hypothesis that cylindrical TJ strands observed in freeze-fracture preparations contain phospholipids.
A histochemical procedure for light microscopic demonstration of xanthine oxidase activity in unfixed cryostat sections using cerium ions and a semipermeable membrane technique.
Authors: Frederiks W M , Marx F
Published: April 2011

Xanthine oxidoreductase exists in two functionally distinct forms. Under normal conditions, the larger part of the enzyme occurs as an NAD(+)-dependent dehydrogenase form which produces NADH and urate. The dehydrogenase can be transformed under various (patho)physiological conditions to an oxygen-dependent oxidase form which produces oxygen radicals and/or hydrogen peroxide and urate. Tetrazolium salts are used to demonstrate the total activity of both the dehydrogenase and the oxidase form of the enzyme. We have developed a procedure to detect the oxidase form only in unfixed cryostat sections with the use of cerium on the basis of the semipermeable membrane technique. The incubation medium contained hypoxanthine as substrate, cerium ions, and sodium azide to inhibit catalase and peroxidase activity. In a second-step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Large amounts of final reaction product were found in milk droplets in the acini of lactating bovine mammary gland, whereas milk-secreting epithelial cells contained hardly any final reaction product. In rat duodenum, enzyme activity was found in the cytoplasm of enterocytes and goblet cells but not in the mucus. Control reactions performed in the absence of substrate or in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase, were completely negative in both tissues, with the exception of polymorphonuclear leukocytes in the lamina propria of duodenum. The positive nonspecific reaction in these cells was caused by myeloperoxidase activity. We conclude that the present method is specific for the detection of xanthine oxidase activity. Moreover, conversion of the dehydrogenase form into the oxidase form can be prevented by omission of chemical fixation of the tissue in the present procedure.
Rapid expression of collagen type X gene of non-hypertrophic chondrocytes in the grafted chick periosteum demonstrated by in situ hybridization.
Authors: Nagamoto N , Iyama K , Kitaoka M , Ninomiya Y , Yoshioka H , Mizuta H , Takagi K
Published: April 2011

We investigated the spatiotemporal localization of collagen Type I, II, and X mRNAs in the subcutaneously grafted chick periosteum by in situ hybridization. Five days after transplantation, we noted three types of histological findings in the grated tissue. (a) Developing trabecular bone exhibited proliferation of spindle-shaped fibroblastic cells and polygonal osteoblasts with moderate signals for collagen Type I mRNA. (b) Developing cartilage contained ovoid chondrocytes with a moderate level of both collagen Type I and II mRNAs. Differentiating chondrocytes with increased collagen Type X mRNA developed during the course of endochondral ossification. (c) An atypical mass of cartilage weakly stained with alcian blue was composed of a large number of non-hypertrophic chondrocytes exhibiting high signals for collagen Type X mRNA. At Day 9, we observed the typical histological features of both membranous and endochondral ossification. However, sparsely distributed chondrocytes with high signals for collagen Type X mRNA were also demonstrated in osteoid and/or woven bone. The phenotype of chondrocytes showing rapid expression of collagen Type X gene derived from grafted periosteum seems to participate in the important role of endochondral bone formation in the early stage of fracture repair.
The histochemical profiles of fast fiber types IIB, IID, and IIA in skeletal muscles of mouse, rat, and rabbit.
Authors: Hämäläinen N , Pette D
Published: April 2011

This study characterized histochemically three fast fiber types (IIB, IID, IIA) in skeletal muscles of mouse, rat, and rabbit, with special reference to fiber types IIB and IID. The results are complemented by biochemical analyses of myosin heavy chain composition in these muscles. Fiber type delineation is based on various methods for mATPase staining with pre-incubations and assays under different conditions. In rat and mouse, IIB and IID fibers can be best distinguished according to their different mATPase stabilities towards formaldehyde and alkaline pH. In rabbit, the method of Matoba and Gollnick using acid pre-incubation provided best and most reproducible results. In addition to their different mATPase stabilities, the three fast fiber types differ with regard to their oxidative capacities and cross-sectional fiber areas in the three species. In general, Type IIB fibers are the largest and least oxidative, Type IIA fibers the smallest and most oxidative, and Type IID fibers intermediate. In rabbit, Type IID fibers are the predominant fast fiber population in extensor digitorum longus, psoas, and tibialis anterior muscles. As judged from histochemistry, these muscles of rabbit do not contain pure Type IIB fibers. This is in accordance with biochemical results that show the HCIId to form the majority of the myosin heavy chain complement expressed in these muscles. On the other hand, IIB fibers are numerous in rabbit adductor magnus, gastrocnemius, and vastus lateralis muscles. Similarly, appreciable amounts of myosin heavy chain HCIIb are found in the three latter muscles of rabbit.
Immunocytochemical identification of basic fibroblast growth factor in the developing rat mammary gland: variations in location are dependent on glandular structure and differentiation.
Authors: Rudland P S , Platt-Higgins A M , Wilkinson M C , Fernig D G
Published: April 2011

We raised antiserum to human recombinant basic fibroblast growth factor (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-growing ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In growing neonatal ducts from 1-day-old rats, in growing terminal end buds (TEBs) and, to a lesser extent, in growing alveolar buds (ABs) in prepubescent (21-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-growing ducts from rats aged 6 days, in non-growing ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-growing ducts, but there is little change in the staining of growing TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in growing structures such as TEBs.
In situ localization of the estrogen receptor in living cells with the fluorescent phytoestrogen coumestrol.
Authors: Miksicek R J
Published: April 2011

Coumestrol is a naturally occurring plant coumarin that displays high affinity for the hormone-binding site of the human estrogen receptor (hER), for which it serves as a potent non-steroidal agonist. Coumestrol emits intense blue fluorescence when bound to this protein, making it ideally suited for use as a cytological stain to detect ER in fixed and intact cells. Conditions are reported for the efficient detection of recombinant ER protein artificially expressed in cultured cells by calcium phosphate-mediated transfection. Coumestrol fluorescence co-localizes with hER protein detected by indirect immunofluorescence with an hER-specific anti-peptide antiserum, confirming the specificity of this reagent. Fluorescence from ER occupied by coumestrol shows a predominantly nuclear distribution, even when the receptor protein is cross-linked in situ by fixation with glutaraldehyde plus paraformaldehyde before coumestrol exposure. This corroborates previous conclusions, based on indirect immunofluorescence analysis, that the ER remains nuclear in the absence of hormone. Examination of the pattern of coumestrol staining in mitotic cells further indicates that hER remains associated with condensed chromatin. Such observations illustrate the potential for using coumestrol to investigate real-time effects of a variety of physiological stimuli on the subcellular distribution of hER in living cells.
Characterization of the phenotype and birthdates of pyknotic dead cells in the nervous system by a combination of DNA staining and immunohistochemistry for 5'-bromodeoxyuridine and neural antigens.
Authors: Soriano E , Del Río J A , Auladell C
Published: April 2011

Cells displaying highly condensed pyknotic nuclei, the most characteristic feature of apoptosis, are considered as dead cells in neural tissue. The present study aimed to devise methods that could allow the neurogenetic and phenotypic characterization of dying pyknotic cells. In the first set of experiments, pregnant mice were labeled at embryonic days E10-E16 with pulses of 5'-bromodeoxyuridine visualization of BrdU after an immunoperoxidase reaction. In addition to normal, healthy immunopositive nuclei, these preparations displayed a number of pyknotic nuclei that were immunoreactive for BrdU. Both the regional and the temporal distribution of BrdU-positive pyknotic cells were coincidental with the peaks of dead cells in neural tissue. For example, pulses of BrdU at E10-E11 resulted in the visualization of immunoreactive pyknotic cells in the subplate and white matter of the cerebral cortex in early postnatal (P) animals. Thus, the times of generation (birthdates) of cells subjected to degenerative processes can be unequivocally identified. In the second set of experiments, brain sections from unlabeled littermates were immunostained for a variety of neural and glial markers and counterstained with bisbenzimide, to find antigens which, by being present in degenerate pyknotic cells, could indicate the phenotype of such cells. Although no pyknotic cells were positively immunostained for neurofilaments, neuropeptide Y, somatostatin, vasoactive intestinal polypeptide, or vimentin, a number of pyknotic cells were found to be immunoreactive for microtubule-associated protein 2, gamma-aminobutyric acid, calbindin 28KD, and glial fibrillary acidic protein. The percentage of pyknotic cells labeled with neural antigens accounted for more than 20% of the total number of pyknotic cells in a given brain region. In contrast, GFAP-positive pyknotic cells represented up to 50% of the total pyknotic cell population. The method shown here has enabled us to determine that both neurons and glial cells undergo degeneration during normal development.
Parenchymal innervation of normal and cirrhotic human liver: a light and electron microscopic study using monoclonal antibodies against the neural cell-adhesion molecule.
Authors: Scoazec J Y , Racine L , Couvelard A , Moreau A , Flejou J F , Bernuau D , Feldmann G
Published: April 2011

Hepatic innervation participates in the control of sinusoidal blood flow and in the regulation of certain metabolic functions of the liver. The study of the distribution of hepatic nerves has been hampered by the lack of adequate markers. We therefore tested the value of the neural cell-adhesion molecule (NCAM) as a probe for the study of parenchymal nerves in the normal and cirrhotic human liver. Four antibodies against various epitopes of NCAM were tested by light and electron microscopic immunohistochemistry: Leu19, ERIC-1, VC1.1, and HNK-1. Their reactivity was compared with that of antibodies against the following neural cell markers: S100 protein, neurofilaments, and neuron-specific enolase (NSE). The tissue reactivity of anti-NCAM antibodies was variable, suggesting a microheterogeneity of the NCAM molecule in the normal liver. Clones Leu19 and ERIC-1 proved to be the most sensitive of the anti-NCAM antibodies. Their sensitivity was superior to that of the antibodies directed against the other neural cell markers tested. In the normal liver, both Leu19 and ERIC-1 demonstrated a heterogeneous distribution of nerve fibers inside the hepatic lobule. Intralobular nerve fibers predominated in Zone 1. This might contribute to the constitution of distinct zonal microenvironments inside the hepatic lobule. In cirrhosis, no nerve fiber was detected inside parenchymal nodules; no nerve plexus was visible at the contact of proliferating neoductules. These alterations might contribute to the pathogenesis of the hemodynamic and metabolic disorders observed in cirrhosis.
Penetration of lucifer yellow into human skin: a lateral diffusion channel in the stratum corneum.
Authors: Mansbridge J N , Knapp A M
Published: April 2011

We investigated the penetration of Lucifer Yellow into human and murine epidermis in 4-mm punch biopsies by incubation in dye solution. Lucifer Yellow was taken up freely by the dermis but penetrated only slightly into keratinocytes of the basal and suprabasal layers. However, progressive lateral diffusion was observed in the lowest layers of the stratum corneum, extending a distance of 1 mm in 6 hr. Under high magnification, Lucifer Yellow appeared to lie within rather than between corneocytes of this layer. Control samples stained with Lucifer Yellow after sectioning showed no preferential binding of the dye in this region. We concluded that the localization of staining was the result of diffusion from the cut edge of the stratum corneum. Lucifer Yellow penetration was insensitive to PMSF, 1:10 phenanthroline, or N-ethyl maleimide and was also observed in an in vivo injury, indicating that it was not an artifact of proteolytic or degenerative changes. In contrast, horseradish peroxidase failed to penetrate, suggesting molecular size limitation to channel entry. Diffusion of Lucifer Yellow beneath the stratum corneum marks a pathway for the lateral movement of small molecules of potential importance in the normal physiology of the skin, drug delivery, and pathology.
In situ blotting: a novel method for direct transfer of native proteins from sectioned tissue to blotting membrane: procedure and some applications.
Authors: Okabe M , Nyakas C , Buwalda B , Luiten P G
Published: April 2011

We describe a novel technique for direct transfer of native proteins from unfixed frozen tissues sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility for molecular detection but retaining the anatomic localization at the cellular level. Within 10 min a maximum protein transfer is achieved independent of the protein molecular weight. The total protein bound was 80% of the maximal binding capacity of the blotting membrane and independent of the section thickness. These results indicate that the proteins that bind to the membrane originate from the cut cell monolayer that has direct contact with the blotting membrane. This in situ blotting method provides direct protein mapping from a single cell layer of a tissue section. The procedure includes cryosectioning at 20 microns and collecting sections on a dry blotting membrane at -20 degrees C. For protein transfer the blotted sections are thawed and incubated for 10 min with Tris buffer. After incubation the sections are removed from the membrane by high-pressure spray. The blotted membranes can be subjected to several detection assays. In the present study the presence of several proteins was demonstrated in brain and thymus by immunochemical and enzyme histochemical procedures.
A fixative suitable for in situ hybridization histochemistry.
Authors: Uehara F , Ohba N , Nakashima Y , Yanagita T , Ozawa M , Muramatsu T
Published: April 2011

We compared the morphology and stability of hybridization signals between paraffin sections of rat retina fixed with commonly used 4% paraformaldehyde/PBS and those fixed with a fixative containing glutaraldehyde in in situ hybridization histochemistry, using a digoxigenin-labeled RNA probe complementary for beta-galactoside alpha 2,6-sialyltransferase mRNA. Retinal detachment was frequently observed in the sections fixed with 4% paraformaldehyde-PBS, whereas the morphology was satisfactorily preserved in those fixed with either 0.5% glutaraldehyde, 4% paraformaldehyde-PBS, or 2.5% glutaraldehyde-PBS. Without glutaraldehyde, it was difficult to determine the most appropriate length of proteinase K digestion of tissue sections for facilitating probe penetration, since the optimal time for definite hybridization was variable among the retinal cells in heterogeneous layers. By addition of glutaraldehyde to paraformaldehyde or with glutaraldehyde alone, it was easy to establish the appropriate time for the unmasking procedure, since intense mRNA signals were constant throughout the retina by proteinase K digestion for more than 30-40 min. Using a fixative that causes stronger cross-linking (e.g., glutaraldehyde) is recommended to improve not only the morphology but also the stability of hybridization signals in in situ hybridization histochemistry with paraffin embedding and digoxigenin-labeled RNA probes.
A modified cerium-based histochemical method for detection of experimentally-induced ATPase impairment in glomeruli of the rat kidney.
Authors: Baller J F , Poelstra K , Hardonk M J , Bakker W W
Published: April 2011

We studied glomerular ATPase activity, as detectable at the light microscopic (LM) level in cryostat sections of the rat kidney, after unilateral local X-irradiation. The biochemically detectable reduction in glomerular ATPase activity after X-irradiation could be demonstrated at the LM level by application of a modified cerium-based technique. Results show a clear reduction of reaction product in glomeruli in X-irradiated kidneys as compared with the contralateral control kidney. Technical parameters (i.e., tissue fixation, second thickness, cerium concentration of the incubation mixture, and percentage H2O2 added for the amplification step) were established for optimal reproducibility of the staining results. We show that this modified staining protocol allows detection of differences of ATPase activity in contrast to conventional histochemical methods. Inhibition studies with various phosphatase inhibitors and competitive substrate inhibition experiments revealed that the enzyme is specific for nucleoside di- and triphosphatases. Since reduced glomerular adenine nucleotidase activity has recently been recognized as an early event in (experimental) glomerulonephritis, we feel that the new staining protocol presented here may be highly relevant for routine tissue section screening in nephropathological research.
Localization of rat tryptase to a subset of the connective tissue type of mast cell.
Authors: Chen Z , Irani A A , Bradford T R , Craig S S , Newlands G , Miller H , Huff T , Simmons W H , Schwartz L B
Published: April 2011

We examined the cellular distribution of rat tryptase in rat skin, lung, small intestine, and peritoneal lavage cells by immunohistochemical techniques. Tryptase purified to apparent homogeneity from rat skin was used to generate a goat polyclonal anti-rat tryptase antibody. Tryptase-containing cells were detected in lung, skin, and peritoneal lavage cells. Small intestine mucosa, on the other hand, showed few if any tryptase-positive cells. Sequential staining with Alcian blue and anti-tryptase antibody showed that tryptase is located only in mast cells. Sequential staining with safranin to identify the connective tissue type of mast cell and anti-tryptase antibody showed that tryptase resides only in this mast cell type. However, only a subpopulation of the safranin-stained mast cells contained tryptase. In lung, 53% of the mast cells stained with safranin; 94% contained tryptase. In skin, 80% stained with safranin; only 6% contained tryptase. In peritoneal cells, more than 95% of the mast cells were stained with safranin; 20% contained tryptase. In the bowel mucosa, where few cells are stained by safranin, no cells with tryptase were detected. The percentages of cells with chymase I that also contained tryptase were 80% and 84% for lung, 4% and 7% for skin, and 15% and 13% for peritoneal cells by respective simultaneous and sequential double labeling with anti-tryptase and anti-chymase I antibodies. This study suggests that the rat connective tissue type of mast cell is subdivided into two forms on the basis of the presence or absence of tryptase, whereas rat mucosal mast cells lack this enzyme. These results contrast with those in humans, in which tryptase is present in all mast cells, but are similar to mice, in which tryptase mRNA has been detected only in the connective tissue type.
Cytoskeletal structure of skeletal muscle: identification of an intricate exosarcomeric microtubule lattice in slow- and fast-twitch muscle fibers.
Authors: Boudriau S , Vincent M , Côté C H , Rogers P A
Published: April 2011

We used immunochemical quantification and indirect immunofluorescence to investigate the cell content, distribution, and organization of microtubules in adult rat slow-twitch soleus and fast-twitch vastus lateralis muscles. An immunoblotting assay demonstrated that the soleus muscle (primarily Type I fibers) was found to have a 1.7-fold higher relative content of alpha-tubulin compared with the superficial portion of the vastus lateralis muscle (primarily Type IIb fibers). Both physiological muscle types revealed a complex arrangement of microtubules which displayed oblique, longitudinal, and transverse orientations within the sarcoplasmic space. The predominance of any one particular orientation varied significantly from one muscle tissue section to another. Nuclei were completely surrounded by a dense net-like structure of microtubules. Both muscle fiber types were found to possess a higher density of microtubules in the subsarcolemmal region. These microtubules followed the contour of the sarcolemma in slightly contracted fibers and showed a fine punctate appearance indicative of a restricted distribution. The immunofluorescence results indicate that microtubules are associated with the sarcolemma and therefore may form a part of the membrane cytoskeletal domain of the muscle fiber. We conclude that the microtubule network of the adult mammalian skeletal muscle fiber constitutes a bone fide component of the exosarcomeric cytoskeletal lattice domain along with the intermediate filaments, and as such could therefore participate in the mechanical integration of the various organelles of the myofibers during the contraction-relaxation cycle.
An improved immunocytochemical staining method for large semi-thin plastic epon sections: application to GABA in rat cerebral cortex.
Authors: Romijn H J , Janszen A W , Pool C W , Buijs R M
Published: April 2011

We describe here a procedure that significantly enhances the intensity and method-specificity of immunocytochemical staining in large mounted semi-thin plastic Epon sections. The procedure was developed for the detection of the neurotransmitter gamma-aminobutyric acid (GABA) in rat brain tissue fixed with glutaraldehyde, but it may also be helpful in unmasking other antigens under different conditions. In addition to some practical suggestions for improving the reproducibility of the staining procedure, we demonstrate that the crucial step in the procedure is pre-treatment of the deplasticized sections with proteinase-K before exposure to the first antibody. This leads to a high morphological resolution and an excellent immunocytochemical signal.
High-resolution imaging of protein phosphorylation in squid axons and synapse by electron energy loss spectroscopy.
Authors: Martin R , Door R , Breitig D
Published: April 2011

We demonstrate that clusters of phosphorus atoms can be detected in energy loss spectroscopic images (ESI) of cytoskeletal proteins of squid axons. In series of images taken at four energy windows below and three windows above the phosphorus P-L2,3 ionization edge, signal-to-background intensity differences were analyzed by videodensitometry. A distinct increase of relative intensities was recorded above the phosphorus edge in neurofilaments of the peripheral giant axon and in those of the presynaptic terminal. A high level of neurofilament phosphorylation in the peripheral axon supports previous biochemical and immunochemical results, but our finding of phosphorylated neurofilaments in the presynaptic axon conflicts with these studies. Our method may be advantageous for analysis with high elemental and spatial resolution of the phosphorylation state of cytoskeletal protein molecules in situ.
Demonstration of anionic sites in human eccrine and apocrine sweat glands in post-embedded ultra-thin sections with cationic colloidal gold: effect of enzyme digestion on these anionic sites.
Authors: Saga K , Takahashi M
Published: April 2011

We localized anionic sites ultrastructurally in human eccrine and apocrine sweat glands with a poly-L-lysine-gold complex (cationic colloidal gold). Anionic sites were labeled by incubating Lowicryl K4M-embedded sections on droplets of cationic colloidal gold. In eccrine sweat glands, colloidal gold particles were restricted to the basolateral membrane of the secretory cells at low pH, whereas the luminal membrane did not react with the gold particles. Chondroitinase ABC digested these anionic sites. This indicates that chondroitin sulfate and/or dermatan sulfate constitutes anionic sites in the basal labyrinth of eccrine sweat glands. In apocrine sweat glands, the luminal membrane of the secretory cells showed strong reaction at low pH, whereas the contraluminal membrane did not show any reaction. Neuraminidase completely digested these anionic sites, which indicated that the anionic charge of the apocrine lumen was due to sialic acid. Differences in distribution and susceptibility to enzymes of anionic sites in cell membranes between eccrine and apocrine sweat glands may reflect functional differences between these glands. Dark cell granules in eccrine secretory cells were negative for the anionic sites when sections were labeled without any pre-treatment. However, pre-incubation of the grids on EGTA or deionized water unmasked the anionic sites on the dark cell granules. The positive staining after EGTA treatment was greatly decreased by reincubation with CaCl2. These results suggested that Ca blocked anionic sites in dark cell granules. Exposed anionic sites were digested with chondroitinase ABC. This indicated that chondroitinase ABC and/or dermatan sulfate composed the anionic sites in dark cell granules.
An intestinal secretory protein is found in most glands associated with the gastrointestinal tract: von Ebner's and salivary glands, gallbladder, and pancreas.
Authors: Calvert R , Millane G , Pothier P , Beaulieu J F
Published: April 2011

We have previously shown that monoclonal antibodies (MAb) prepared against the duodenal mucosa of 4-day-old mice disclosed the presence of two antigens associated with the formation of intestinal crypts. One of these, MIM-1/39, was found in the apical cytoplasm of undifferentiated epithelial crypt cells of the duodenum and colon. We report here the immunolocalization of MIM-1/39 in different glands associated with the gastrointestinal tract, using a polyclonal antibody produced against antigen MIM-1/39. By indirect immunofluorescence on 1-micron thick Lowicryl K4M sections, MIM-1/39 was detected in secretory granules of serous cells in lingual (von Ebner's gland), sublingual, submandibular and parotid glands, and in pancreas; it was also found in epithelial cells of the gallbladder and in the secretory granules of chief cells of gastric glands. Liver, kidney, and Brunner's glands were not immunoreactive. Immunocytochemistry revealed the presence of antigen MIM-1/39 in small secretory granules of the gallbladder, duodenum, colon, and in the large secretory granules in serous cells of lingual and parotid glands, in pancreas, and in gastric chief cells. In Western blotting, the MIM-1/39 MAb revealed two bands (330 and 350 KD) in adult mouse duodenal mucosa, gallbladder and stomach, whereas only one (330 KD) was disclosed in pancreatic juice. However, two bands (330 and 350 KD) were detected in pancreatic juice with the polyclonal antibody. The distribution of MIM-1/39 was different from that reported for IgA, bound and free secretory components, cryptdin, and Tamm-Horsfall protein. Therefore, MIM-1/39 appears to be a unique protein. Its exact role remains to be elucidated.
Immunoelectron microscopy: a new method for detection of insulin antibodies.
Authors: Saccomanno K , Taccagni G , Bosi E , Preti P , Dozio N , Cantaboni A
Published: April 2011

The aim of the present study was to set up a sensitive technical alternative to the classical procedures for detection of human insulin antibodies. We developed a method of post-embedding immunoelectron microscopy (IEM) using as the substrate fresh human pancreas, embedded in acrylic resin to maintain its antigenic structure. The antigen was insulin within the mature secretory granule. Serum samples obtained from 10 patients with insulin antibodies detected at various titers by either radio binding assay (RBA) or enzymatic immunoassay (EIA) were incubated for 2 hr at 37 degrees C at dilutions of 1:25, 1:100, 1:400, 1:1600, 1:6400, 1:25,600, and 1:102,400. The electron microscope photographs were analyzed by computerized morphometry and the number of protein A-gold-IgG complexes was calculated per micron2 of insulin granule. IEM results were compared with those obtained with EIA. The specificity of both techniques towards insulin was assessed as the difference between the signals (number of gold particles per micron2 of insulin granule in IEM or optical density > or = 0.193 in EIA) with and without excess insulin. Sensitivity was defined as the detection limit of the assay. In all the 10 sera investigated, IEM was more sensitive, with a 12- to 40-fold lower detection limit than EIA. IEM, with native insulin granules as substrate, is a specific, reproducible, and sensitive method for detection of human serum insulin antibodies. These findings also suggest IEM as a procedure potentially suitable for identifying antigen specificity of autoantibodies circulating at low concentration.
Flow cytometric sorting of living, highly motile human spermatozoa based on evaluation of their mitochondrial activity.
Authors: Auger J , Leonce S , Jouannet P , Ronot X
Published: April 2011

We investigated the applicability of flow cytometric (FCM) sorting to select, with no deleterious effects, fractions of living, highly motile spermatozoa after staining with rhodamine 123 (Rh123) and propidium iodide (PI) for assessment of their mitochondrial activity and viability, respectively. Sperm cells were subjected to FCM sorting according to their Rh123 fluorescence intensity, and computer-aided sperm analysis (CASA) for percentage motility and movement characteristic measurements was carried out on the entire sperm populations and on the Rh123-positive (Rh123+) sorted fractions. A first experiment on five sperm samples from fertile donors pre-selected by either swim-up or simplified Percoll gradient indicated no detrimental effect of the FCM sorting procedure because: (a) the numbers of Rh123+ motile sperm were not decreased by FCM sorting; (b) data on the sorted fractions showed a tendency (not significant) for an increase in movement parameters rather than a drop; and (c) a significant decrease in the percentage of PI-positive (PI+) sperm (13% vs 3%; p < 0.05) was measured. A second experiment was performed on sperm samples from four patients, only washed and re-suspended in B2 medium. This demonstrated a significant increase in some characteristics of movement quality related to a substantial and selective immobilization of the less motile sperm. The significant drop in the percentage of PI+ sperm after FCM sorting (p < 0.01) was less pronounced than after FCM sorting of pre-selected sperm (12% vs 3%, respectively), indicating a lethal effect of FCM sorting on a small proportion of presumably moribund sperm. These preliminary data indicate a differential effect of FCM sorting on sperm according to their function characteristics and suggest the potential importance of these methods for the characterization in vitro of sperm subpopulations on the basis of functional criteria.
Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.
Authors: Fleischmajer R , MacDonald E D , Contard P , Perlish J S
Published: April 2011

Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.
A histological processing technique that preserves the integrity of calcified tissues (bone, enamel), yolky amphibian embryos, and growth factor antigens in skeletal tissue.
Authors: Bourque W T , Gross M , Hall B K
Published: April 2011

We have devised a processing technique to embed calcified tissues, such as bone and tooth enamel, in paraffin, to preserve the delicate antigenic sites of molecules such as growth factors. The same technique, omitting the decalcification step, allows delicate tissues, such as axolotl embryos (Ambystoma mexicanum) containing large yolk masses, to be easily handled during tissue processing and to be serially sectioned. Specimens were all fixed in periodate-lysine-paraformaldehyde (PLP) fixative at 5 degrees C. Bone and teeth were decalcified in an EDTA-G solution at -4 degrees C. Maintaining a temperature of 5 degrees C, the decalcified samples were then washed (with PBS, pH 7.2, under vacuum) to remove glycerol. Both the decalcified tissues and the yolky embryos were dehydrated through an ascending series of isopropanol and embedded in low melting-point paraffin under vacuum. Acidic fibroblast growth factor (aFGF) was located in cells of the expanded cambial layer in the early fracture calluses of male CD-1 mice, demonstrating retention of antigenic sites. The results reported here have not previously been obtained with existing processing and embedding techniques.
In vitro autoradiographic visualization of occupied estrogen receptors in the rat brain with an iodinated estrogen ligand.
Authors: Walters M J , Brown T J , Hochberg R B , MacLusky N J
Published: April 2011

Methods have been developed for the selective measurement of occupied estrogen receptors (ER) in brain tissue sections. Cryostat sections of unfixed tissue were incubated with radiolabeled estrogen at physiological temperatures, displacing endogenous receptor-bound estrogen by radioligand and thereby allowing the receptor complexes to be visualized autoradiographically after washing to remove nonspecifically bound steroid. The resultant autoradiographs were analyzed by computer-assisted densitometry. Synthetic 11 beta-methoxy-substituted radiolabeled estrogens gave the best autoradiographic images, as a result of reduced nonspecific labeling, although [3H]-estradiol was also used successfully. With the synthetic ER ligand 11 beta-methoxy 16 alpha-[125I]-iodo-estradiol, exposure times of less than 24 hr generated acceptable autoradiographs; with 3H-labeled estrogens, exposures of 3 months or more may be required. The method is sufficiently sensitive to detect physiological changes in ER occupation and to allow determination of receptor affinities and saturation binding capacities in discrete cell groups identified in sections from individual animals.
Secretion of chondroitin sulfate from embryonic epidermal cells in Xenopus laevis.
Authors: Nishikawa S , Sasaki F
Published: April 2011

We examined proteoglycans (PGs) in amphibian epidermal cells by immunofluorescence microscopy. Immunoelectron microscopy with pre- and post-embedding methods, combined with HRP- or gold-conjugated secondary antibody, revealed ultrastructural localization of glycosaminoglycans (GAGs). Embryonic epidermis secretion granules in Xenopus laevis contained chondroitin 6-sulfate and unsulfated chondroitin. Immature secretion granules were also labeled with anti-chondroitin 6-sulfate and anti-chondroitin. A step-wise digestion experiment on chondroitinase ABC revealed that fine filaments in the secretion granules were chondroitin sulfate chains. Inhibition experiments with brefeldin A revealed that the life of a secretion granule was 5-10 hr, suggesting that GAG secretion from embryonic epidermis is routed through a regulated pathway.
Expression of decorin mRNA in the nervous system of rat.
Authors: Hanemann C O , Kuhn G , Lie A , Gillen C , Bosse F , Spreyer P , Müller H W
Published: April 2011

A rat cDNA clone (pCD67) isolated from a cDNA library of regenerating sciatic nerve by differential hybridization screening revealed 75% homology on the nucleic acid level and 81% homology (including conservative amino acid changes) to the deduced amino acid sequence of the core protein of human dermatan/chondroitin sulfate proteoglycan decorin (PGII, PG40, PG-S2). Two transcripts of 1.3 and 1.75 KB very similar in size to the two decorin mRNA species previously identified in connective tissue were detected by Northern blotting in both normal and injured sciatic nerve and in the mature and embryonic rat brain. The steady-state level of the decorin 1.3 KB mRNA was very much higher in peripheral nerve than in the central nervous system or in other non-neural tissues (skeletal muscle, heart, colon, kidney). In situ hybridization experiments indicated that decorin mRNA is expressed by Schwann cells and vascular cells in peripheral nerve. In the spinal cord the ventral horn motor neurons and other neurons in gray matter showed specific hybridization signals. Furthermore, in situ hybridization indicated decorin expression in Purkinje neurons and cells of the molecular layer in cerebellum, and in neurons of the primary olfactory cortex and brainstem (pons). Our data clearly demonstrate decorin mRNA expression in distinct neural cell populations, suggesting yet unknown functions of this proteoglycan in the peripheral and central nervous system.
Modulation of retinoblastoma cell characteristics by hexamethylene bis-acetamide and other differentiating agents in culture.
Authors: Rajagopalan S , Rodrigues M , Polk T , Wilson D , Chader G J , Hayden B J
Published: April 2011

The undifferentiated Y-79 retinoblastoma cell line can be induced by specific agents to express characteristics of mature retinal cells. In the present study, attached Y-79 cell cultures were treated with hexamethylene bis-acetamide (HMBA) and other differentiating agents and examined for "neuronal" and other properties. Immunocytochemical staining was performed with antibodies against neuron- and retina-specific antigens, [synaptophysin, interphotoreceptor retinoid-binding protein (IRBP), neural cell adhesion molecule (N-CAM), and rod- and cone-specific transducin (TR alpha and TC alpha)] and microtubule-associated protein (MAP-1) and tubulin. Enhanced expression of tubulin was observed with cAMP treatment in FBS media. Expression of N-CAM was observed in all groups. Morphological differentiation was pronounced with HMBA and butyrate treatment, with HMBA inducing increased tubulin expression after 2 weeks of treatment. Expression of TR alpha was minimal under all culture conditions, whereas TC alpha was ubiquitously expressed. This supports the concept that Y-79 retinoblastoma is predominantly of cone neuronal origin and that, surprisingly, immunocytochemical differentiation is not correlated with the marked morphological changes induced by the major differentiating agents used.
Rapid purification of glycosyl-phosphatidylinositol-anchored alkaline phosphatase from human neutrophils after up-regulation to the cell surface.
Authors: Cain T J , Liu Y , Kobayashi T , Robinson J M
Published: April 2011

Alkaline phosphatase (APase) belongs to a growing family of membrane-associated proteins tethered to the lipid bilayer via a glycosyl-phosphatidylinositol (GPI) anchor. Human neutrophils contain an intracellular pool of APase associated with a novel membrane-bound compartment. Stimulation of neutrophils with the chemotactic peptide formyl-Met-Leu-Phe (fMLP) leads to rapid up-regulation of essentially all of the APase to sites in continuity with the extracellular medium. Pre-treatment of neutrophils with cytochalasin B (cyto B) followed by fMLP likewise leads to expression of the enzyme on the cell surface and a dramatic alteration in cell morphology, but subsequent internalization of the plasmalemma is minimized. Pre-treatment with cyto B and fMLP has been used for isolation and purification of neutrophil APase. Specifically, neutrophils were treated with phosphatidylinositol-specific phospholipase C to release GPI-anchored proteins from the cell surface. APase was purified from supernatants of these preparations by electrophoresis in a non-denaturing gel system and subsequent electroelution. With this approach we rapidly purified neutrophil APase to homogeneity; this protein was then used for immunization. Immunoblotting, ELISA, and immunocytochemical localization were used to characterize the resulting antibodies.
A new approach to map transcription sites at the ultrastructural level.
Authors: Testillano P S , Gorab E , Risueño M C
Published: April 2011

We describe a new ultrastructural method for locating transcription on ultra-thin sections. The use of anti-DNA/RNA hybrid antibodies provides specific labeling on precise structures of the nuclear compartments of several cell types. All mammalian and plant material studied (HeLa cells, lymphocytes, onion root meristematic cells) showed the same pattern of labeling: fibrillar structures in the interchromatin region and discrete regions of the dense fibrillar component at the periphery of the fibrillar centers in the nucleolus. The specificity of the immunogold labeling was tested by RNAse H digestion and by pre-blocking the antibody with synthetic DNA/RNA hybrids; in both cases no gold particles were observed. This method has considerable advantages compared with current techniques, constituting a very useful tool to map transcriptionally active loci in a variety of cells.