| 1. |
- Ackermann, Paul, et al.
(författare)
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An opioid system in connective tissue : A study of Achilles tendon in the rat
- 2001
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 49:11, s. 1387-1395
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Tidskriftsartikel (refereegranskat)abstract
- The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, delta -opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [H-3]-naloxone (K-d 7.01 +/- 0.98 nM; B-max 23.52 +/- 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery.
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| 2. |
- Aitola, M, et al.
(författare)
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Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis
- 2003
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Ingår i: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. - : SAGE Publications. - 0022-1554. ; 51:4, s. 455-469
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Tidskriftsartikel (refereegranskat)abstract
- Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop–helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.
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| 3. |
- Akner, Gunnar, 1953-, et al.
(författare)
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Morphometric studies of the localization of the glucocorticoid receptor in mammalian cells and of glucocorticoid hormone-induced effects
- 1994
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 42:5, s. 645-657
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Tidskriftsartikel (refereegranskat)abstract
- We studied the subcellular distribution of the glucocorticoid receptor (GR) by light microscopy (LM) and confocal laser scanning microscopy (CLSM) in different mammalian cell types. The effect of added glucocorticoid hormones on GR distribution was investigated by photometric quantitation on optical sections obtained by CLSM followed by statistical analysis. In the control interphase cytoplasm, the distribution of GR was fibrillar in some and diffuse in other cell types. Fibrillar GR was distributed along cytoplasmic microtubules (MTs) with predilection for a subset of MTs. GR was also observed in the centrosomes. Nuclear GR was both diffuse and granular in distribution. During cell division, GR appeared in the mitotic apparatus at all stages of mitosis. These findings were not fixation-dependent. Glucocorticoid treatment increased both the nuclear and cytoplasmic GR signal. However, this was detectable only after precipitating but not cross-linking fixation. There was both intra- and intercellular GR heterogeneity in the absence and presence of hormone but no indication of a hormone-induced nuclear translocation of GR. We present a hypothetical model of two independent GR populations in the nucleus and cytoplasm, respectively, without any discernible ligand-induced nuclear translocation of GR. The extranuclear GR population may exert effect(s) on site in the cytoplasm without involving nuclear genomic transcription.
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| 4. |
- Al-Khalili Szigyarto, Cristina, et al.
(författare)
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The E3 Ligase Axotrophin/MARCH-7 : Protein Expression Profiling of Human Tissues Reveals Links to Adult Stem Cells
- 2010
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 58:4, s. 301-308
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Tidskriftsartikel (refereegranskat)abstract
- Axotrophin/MARCH-7 was first identified in mouse embryonic stem cells as a neural stem cell gene. Using the axotrophin/MARCH-7 null mouse, we discovered profound effects on T lymphocyte responses, including 8-fold hyperproliferation and 5-fold excess release of the stem cell cytokine leukemia inhibitory factor (LIF). Our further discovery that axotrophin/MARCH-7 is required for targeted degradation of the LIF receptor subunit gp190 implies a direct role in the regulation of LIF signaling. Bioinformatics studies revealed a highly conserved RING-CH domain in common with the MARCH family of E3-ubiquitin ligases, and accordingly, axotrophin was renamed "MARCH-7." To probe protein expression of human axotrophin/MARCH-7, we prepared antibodies against different domains of the protein. Each antibody bound its specific target epitope with high affinity, and immunohistochemistry cross-validated target specificity. Forty-eight human tissue types were screened. Epithelial cells stained strongly, with trophoblasts having the greatest staining. In certain tissues, specific cell types were selectively positive, including neurons and neuronal progenitor cells in the hippocampus and cerebellum, endothelial sinusoids of the spleen, megakaryocytes in the bone marrow, crypt stem cells of the small intestine, and alveolar macrophages in the 7 lung. Approximately 20% of central nervous system neuropils were positive. Notably, axotrophin/MARCH-7 has an expression profile that is distinct from that of other MARCH family members. This manuscript contains online supplemental material at http://www.jhc. org. Please visit this article online to view these materials. (J Histochem Cytochem 58:301-308, 2010)
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| 5. |
- Almqvist, PM, et al.
(författare)
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Immunohistochemical detection of nestin in pediatric brain tumors
- 2002
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Ingår i: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. - : SAGE Publications. - 0022-1554. ; 50:2, s. 147-158
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Tidskriftsartikel (refereegranskat)abstract
- Nestin is an intermediate filament protein (IFP) expressed in undifferentiated cells during CNS development and in CNS tumors. Previous studies have arrived at different conclusions in terms of which types of CNS tumors express nestin. In this report we establish an immunohistochemical protocol using antigen retrieval, which significantly enhances staining with two polyclonal anti-nestin antisera, #130 and #4350. The staining pattern was identical for the two nestin antisera and very similar to that of vimentin, while glial fibrillary acidic protein (GFAP), immunoreactivity was absent from 9.5-week-old forebrain. The current study of 20 primary CNS tumors from pediatric patients included seven ependymomas, seven primitive neuroectodermal tumors (PNETs), five pilocytic astrocytomas, and one glioblastoma multiforme (GBM). All these tumors expressed nestin to various extents, in contrast to five brain metastases tested. Strong nestin immunoreactivity was found in malignant primary CNS tumors, whereas benign pilocytic astrocytomas showed low but consistent nestin expression. In all tumors nestin immunoreactivity was confined to the cytoplasm of tumor cells and was co-expressed with astrocyte markers vimentin, GFAP, and S-100. Vascular endothelial cells of all neoplasms also showed marked immunoreactivity for nestin and vimentin, whereas they were negative for GFAP and S-100. In conclusion, antiserum #4350 detected nestin in formalin-fixed, paraffin-embedded tissue sections by heat-induced antigen retrieval immunohistochemistry. Nestin was expressed in both highly malignant and low malignant gliomas, indicating the potential use of nestin as a diagnostic tumor marker in surgical pathology.
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| 6. |
- Andersson, Ann-Catrin, et al.
(författare)
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Analysis of protein expression in cell microarrays : A tool for antibody-based proteomics
- 2006
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Ingår i: Journal of Histochemistry and Cytochemistry. - 0022-1554 .- 1551-5044. ; 54:12, s. 1413-1423
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Tidskriftsartikel (refereegranskat)abstract
- Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TIVIA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.
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| 7. |
- Andersson, Sandra, et al.
(författare)
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Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection
- 2013
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 61:11, s. 773-784
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-based protein profiling on a global scale using immunohistochemistry constitutes an emerging strategy for mapping of the human proteome, which is crucial for an increased understanding of biological processes in the cell. Immunohistochemistry is often performed indirectly using secondary antibodies for detection, with the benefit of signal amplification. Direct immunohistochemistry instead brings the advantage of multiplexing; however, it requires labeling of the primary antibody. Many antibody-labeling kits do not specifically target IgG and may therefore cause labeling of stabilizing proteins present in the antibody solution. A new conjugation method has been developed that utilizes a modified Z-domain of protein A (ZBPA) to specifically target the Fc part of antibodies. The aim of the present study was to compare the ZBPA conjugation method and a commercially available labeling kit, Lightning-Link, for in situ protein detection. Fourteen antibodies were biotinylated with each method and stained using immunohistochemistry. For all antibodies tested, ZBPA biotinylation resulted in distinct immunoreactivity without off-target staining, regardless of the presence of stabilizing proteins in the buffer, whereas the majority of the Lightning-Link biotinylated antibodies displayed a characteristic pattern of nonspecific staining. We conclude that biotinylated ZBPA domain provides a stringent method for antibody biotinylation, advantageous for in situ protein detection in tissues.
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| 8. |
- Berggård, Tord, et al.
(författare)
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Alpha1-microglobulin is found both in blood and in most tissues
- 1998
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Ingår i: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society. - : SAGE Publications. - 0022-1554. ; 46:8, s. 94-887
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Tidskriftsartikel (refereegranskat)abstract
- In this study we demonstrate that, in addition to blood, alpha1-microglobulin (alpha1m) is present in most tissues, including liver, heart, eye, kidney, lung, pancreas, and skeletal muscle. Western blotting of perfused and homogenized rat tissue supernatants revealed alpha1m in its free, monomeric form and in high molecular weight forms, corresponding to the complexes fibronectin-alpha1m and alpha1-inhibitor-3-alpha1m, which have previously been identified in plasma. The liver also contained a series of alpha1m isoforms with apparent molecular masses between 40 and 50 kD. These bands did not react with anti-inter-alpha-inhibitor antibodies, indicating that they do not represent the alpha1m-bikunin precursor protein. Similarly, the heart contained a 45-kD alpha1m band and the kidney a 50-kD alpha1m band. None of these alpha1m isoforms was present in plasma. Immunohistochemical analysis of human tissue demonstrated granular intracellular labeling of alpha1m in hepatocytes and in the proximal epithelial cells of the kidney. In addition, alpha1m immunoreactivity was detected in the interstitial connective tissue of heart and lung and in the adventitia of blood vessels as well as on cell surfaces of cardiocytes. alpha1m mRNA was found in the liver and pancreas by polymerase chain reaction, suggesting that the protein found in other tissues is transported via the bloodstream from the production sites in liver and pancreas. The results of this study indicate that in addition to its role in plasma, alpha1m may have important functions in the interstitium of several tissues. (J Histochem Cytochem 46:887-893, 1998)
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| 9. |
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| 10. |
- Brismar, Hjalmar, et al.
(författare)
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Spectra and fluorescence lifetimes of lissamine rhodamine, tetramethylrhodamine isothiocyanate, texas red, and cyanine 3.18 fluorophores : influences of some environmental factors recorded with a confocal laser scanning microscope.
- 1995
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Ingår i: Journal of Histochemistry and Cytochemistry. - : SAGE Publications. - 0022-1554 .- 1551-5044. ; 43:7
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Tidskriftsartikel (refereegranskat)abstract
- We report on the spectra and fluorescence lifetimes of four commonly used fluorophores: lissamine rhodamine (LRSC); tetramethyl rhodamine isothiocyanate (TRITC); Texas Red; and cyanine 3.18 (Cy-3). Fluorescence lifetime recordings revealed that these spectrally overlapping fluorophores can be individually detected by their lifetimes, indicating that at least four fluorophores can be individually identified in discrete tissue domains by confocal microscopy. A further advantage of lifetime recordings is that fluorophores that emit light within the same wavelength band can be used and chromatic aberrations are therefore circumvented, thereby improving the spatial accuracy in imaging of multiple fluorophores. Low and high pH, respectively, tended to influence fluorophore emission spectra and fluorescence lifetime. IgG conjugation of the fluorophores tended to shift the spectra towards longer wavelengths and to change the fluorescence lifetimes. The IgG-conjugated form of the fluorophores may, when applied to tissue specimens, change the emission spectrum and lifetime. In addition, different tissue embedding procedures may influence fluorescence lifetime. These observations emphasize the importance of spectral and lifetime characterization of fluorescent probes within the chemical context in which they will be used experimentally. Changes in spectra and fluorescence lifetimes may be a useful tool to gain information about the chemical environment of the fluorophores.
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