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  • Detection of UCH-L1 expression by pre-embedding immunoelectron microscopy with colloidal gold labeling in diseased glomeruli. 18300032

    The purpose of this report was to study the use of pre-embedding immunoelectron microscopy technique with gold and horseradish peroxidase (HRP) labeling in detecting the expression of ubiquitin C-terminal hydrolase 1 (UCH-L1) of podocytes in glomerulonephritis. The specimens of human IgA nephropathy and lupus nephritis were fixed with paraformaldehyde and lysine-HCl buffer, labeled by colloidal gold or HRP, embedded with epoxy resin, and examined under the transmission electron microscope. The high density of gold particles or peroxidase reaction products (DAB) combined with UCH-L1 was obvious in cytoplasm and processes of podocytes. This modified technique of pre-embedding immunoelectron microscopy could perfectly preserve the ultrastructure of kidney and expose antigens which is valuable for clinical diagnostic work and experimental research.
    Document Type:
    Reference
    Product Catalog Number:
    AB1761

  • Occurrence and phase distribution of selected pharmaceuticals in the Yangtze Estuary and its coastal zone. 21497014

    The occurrence and geochemical behavior of nine pharmaceutical compounds were investigated along the Yangtze River Estuary and its coastal area, by sampling and analysis of pharmaceuticals in sediment, suspended particulate matter (SPM), colloidal and soluble phases. In addition, the impact of sewage input was examined by sampling from sewage treatment plants (STP) effluent and its upstream and downstream in the Yangtze River. Although at relatively low concentrations in SPM and sediments, several pharmaceuticals were found at elevated concentration in filtered water samples from STP-affected sites. STP is therefore an important input of pharmaceuticals in the study area. Colloidal phase was further separated from bulk water samples using cross-flow ultrafiltration (CFUF), confirming it being an effective sorbent for pharmaceuticals with high sorption capacity which are 2-4 orders of magnitude higher than SPM. Moreover, mass balance calculations showed that significant percentages of selected pharmaceutical compounds were associated with aquatic colloids, indicating colloids as a reservoir for these contaminants in the Yangtze estuarine system.Copyright © 2011 Elsevier B.V. All rights reserved.
    Document Type:
    Reference
    Product Catalog Number:
    16-103
    Product Catalog Name:
    Anti-Phosphotyrosine Antibody, clone 4G10®, Biotin Conjugate

  • Nanoparticle targeting to neurons in a rat hippocampal slice culture model. 22973864

    We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP(9)GGH(6) (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Motor axon synapses on renshaw cells contain higher levels of aspartate than glutamate. 24816812

    Motoneuron synapses on spinal cord interneurons known as Renshaw cells activate nicotinic, AMPA and NMDA receptors consistent with co-release of acetylcholine and excitatory amino acids (EAA). However, whether these synapses express vesicular glutamate transporters (VGLUTs) capable of accumulating glutamate into synaptic vesicles is controversial. An alternative possibility is that these synapses release other EAAs, like aspartate, not dependent on VGLUTs. To clarify the exact EAA concentrated at motor axon synapses we performed a quantitative postembedding colloidal gold immunoelectron analysis for aspartate and glutamate on motor axon synapses (identified by immunoreactivity to the vesicular acetylcholine transporter; VAChT) contacting calbindin-immunoreactive (-IR) Renshaw cell dendrites. The results show that 71% to 80% of motor axon synaptic boutons on Renshaw cells contained aspartate immunolabeling two standard deviations above average neuropil labeling. Moreover, VAChT-IR synapses on Renshaw cells contained, on average, aspartate immunolabeling at 2.5 to 2.8 times above the average neuropil level. In contrast, glutamate enrichment was lower; 21% to 44% of VAChT-IR synapses showed glutamate-IR two standard deviations above average neuropil labeling and average glutamate immunogold density was 1.7 to 2.0 times the neuropil level. The results were not influenced by antibody affinities because glutamate antibodies detected glutamate-enriched brain homogenates more efficiently than aspartate antibodies detecting aspartate-enriched brain homogenates. Furthermore, synaptic boutons with ultrastructural features of Type I excitatory synapses were always labeled by glutamate antibodies at higher density than motor axon synapses. We conclude that motor axon synapses co-express aspartate and glutamate, but aspartate is concentrated at higher levels than glutamate.
    Document Type:
    Reference
    Product Catalog Number:
    AB1588

  • Type VI collagen in extracellular, 100-nm periodic filaments and fibrils: identification by immunoelectron microscopy. 3525575

    Filaments and fibrils that exhibit a 100-nm axial periodicity and occur in the medium and in the deposited extracellular matrix of chicken embryo and human fibroblast cultures have been tentatively identified with type VI collagen on the basis of their similar structural characteristics (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Using indirect immunoelectron microscopy and specific monoclonal and polyclonal antibodies, we now report their positive identification with collagen VI and their distribution in fibroblast cultures and in tendon. Primary human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, showed a progressive increase in labeling and changes in distribution with time up to 8 d in culture. With immunoelectron microscopy and monoclonal antibodies to human type VI collagen followed by goat anti-mouse IgG coupled to colloidal gold, they showed in thin sections specific 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) attached to the band region and another (4B10) to the interband region of the filaments and fibrils. Rabbit antiserum to type VI collagen also localized on the band region, but the staining was less well defined. Control experiments with antibodies to fibronectin and to procollagen types I and III labeled other filaments and fibrils, but not those with a 100-nm period. Heavy metal-stained fibrils with the same periodic and structural characteristics also have been found in both adult rat tail tendon and embryonic chicken tendon subjected to prolonged incubation in culture medium or treatment with adenosine 5'-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils represent the native aggregate form of type VI collagen. It is likely that banded fibrils of the same periodicity and appearance, reported by many observers over the years in a wide range of normal and pathological tissues, are at least in part, type VI collagen.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1944
    Product Catalog Name:
    Anti-Collagen Type VI Antibody, clone 3C4

  • Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody against slow myosin. 3279166

    Existing methods for the ultrastructural identification of fibre types in human skeletal muscle are fallible. This has prompted us to develop a reliable immunoelectron microscopic approach for the identification of human skeletal muscle fibre types. Here we report the unambiguous electron microscopic identification of human type 1 muscle fibres, achieved by combining cryoultramicrotomy with colloidal gold immunocytochemical labelling, using a monoclonal antibody (N0Q7.5.4D) which is specific for the heavy chain of the slow myosin isoform of human skeletal muscle. This method for the identification of muscle fibre types and determination of myosin isoform distributions may have important applications in the ultrastructural study of pathological muscle and in the analysis of myofibrillar assembly during myogenesis.
    Document Type:
    Reference
    Product Catalog Number:
    MAB1628
    Product Catalog Name:
    Anti-Myosin Antibody, slow muscle, clone NOQ7.5.4D

  • Detergent resistant membrane-associated IDE in brain tissue and cultured cells: Relevance to Abeta and insulin degradation. 19117523

    Insulin degrading enzyme (IDE) is implicated in the regulation of amyloid beta (Abeta) steady-state levels in the brain, and its deficient expression and/or activity may be a risk factor in sporadic Alzheimer's disease (AD). Although IDE sub-cellular localization has been well studied, the compartments relevant to Abeta degradation remain to be determined.Our results of live immunofluorescence, immuno gold electron-microscopy and gradient fractionation concurred to the demonstration that endogenous IDE from brain tissues and cell cultures is, in addition to its other localizations, a detergent-resistant membrane (DRM)-associated metallopeptidase. Our pulse chase experiments were in accordance with the existence of two pools of IDE: the cytosolic one with a longer half-life and the membrane-IDE with a faster turn-over. DRMs-associated IDE co-localized with Abeta and its distribution (DRMs vs. non-DRMs) and activity was sensitive to manipulation of lipid composition in vitro and in vivo. When IDE was mis-located from DRMs by treating cells with methyl-beta-cyclodextrin (MbetaCD), endogenous Abeta accumulated in the extracellular space and exogenous Abeta proteolysis was impaired. We detected a reduced amount of IDE in DRMs of membranes isolated from mice brain with endogenous reduced levels of cholesterol (Chol) due to targeted deletion of one seladin-1 allele. We confirmed that a moderate shift of IDE from DRMs induced a substantial decrement on IDE-mediated insulin and Abeta degradation in vitro.Our results support the notion that optimal substrate degradation by IDE may require its association with organized-DRMs. Alternatively, DRMs but not other plasma membrane regions, may act as platforms where Abeta accumulates, due to its hydrophobic properties, reaching local concentration close to its Km for IDE facilitating its clearance. Structural integrity of DRMs may also be required to tightly retain insulin receptor and IDE for insulin proteolysis. The concept that mis-location of Abeta degrading proteases away from DRMs may impair the physiological turn-over of Abeta in vivo deserves further investigation in light of therapeutic strategies based on enhancing Abeta proteolysis in which DRM protease-targeting may need to be taken into account.
    Document Type:
    Reference
    Product Catalog Number:
    AB5352
    Product Catalog Name:
    Anti-Amyloid Precursor Protein Antibody, CT

  • Dissociated secondary hyperalgesia in a subject with a large-fibre sensory neuropathy. 8393170

    In the skin surrounding a site of injury, hyperalgesia develops to mechanical stimuli. Two types of secondary hyperalgesia (to light touch and punctate stimuli) have recently been differentiated, based on different durations and sizes of the area involved. We studied secondary hyperalgesia in a subject who had a loss of myelinated afferent nerve fibres below the neck that spared the A delta group. Stroking with a cotton swab was not perceived anywhere on affected skin either before or after injection of 60 micrograms of capsaicin. Thus, there was no hyperalgesia to light touch. Capsaicin injection into the volar forearm evoked normal pain and flare. A von Frey probe exerting a force of 40 mN was perceived as sharp. The sensation of sharpness was more pronounced up to 2 cm outside the flare zone for at least 16 min following the injection (tested with a 200 mN von Frey probe). Thus, hyperalgesia to punctate stimuli developed as in healthy subjects. These data support the model that hyperalgesia to light touch (allodynia) is due to sensitisation of central pain-signaling neurones to low-threshold mechanoreceptor input (A beta fibres). In contrast, punctate hyperalgesia is likely to be due to sensitisation to nociceptor input (A delta or C fibres).
    Document Type:
    Reference
    Product Catalog Number:
    20-119

  • Observations on the expression of human papillomavirus major capsid protein in HeLa cells. 26064080

    The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells.HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control.HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells.The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.
    Document Type:
    Reference
    Product Catalog Number:
    MAB837
    Product Catalog Name:
    Anti-Papillomavirus Antibody, 1, 6, 11, 16, 18, 31, clone 1H8

  • Localization of immunoreactive gonadotropin-releasing hormone and relative expression of its mRNA in the oviduct during pregnancy in rats. 17283369

    This study was designed to determine the cellular and ultrastructural distribution of the gonadotropin-releasing hormone (GnRH) and the relative expression of its mRNA in the oviduct of rats during different time points (days 7, 9, 16, and 20) of pregnancy. Immunofluorescent localization and confocal microscopic techniques were used to determine the cellular distribution of GnRH in the oviduct. Immunogold electron microscopy indicated its localization at the ultrastructural level, and real-time PCR was used to study the expression pattern of GnRH mRNA in the oviduct during pregnancy. In general, GnRH was localized within the epithelial cells lining the oviductal lumen at each selected time point. A strong correlation between the fluorescence intensity of GnRH-immunoreactive cells and the relative expression of GnRH mRNA was noted on days 7 and 16, followed by a plateau by day 20. At the ultrastructural level, uniform labeling of colloidal gold particles was observed in secretory vesicles and lamella of the luminal epithelium as well as the lumen of the oviduct. Collectively, these results demonstrate for the first time that the oviductal epithelium synthesizes and secretes the decapeptide GnRH during pregnancy in rats, which may have a possible role in postimplantation embryonic development and the maintenance of pregnancy.
    Document Type:
    Reference
    Product Catalog Number:
    MAB5456