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Immunohistochemical Experiments (immunohistochemical + experiment)
Selected AbstractsA novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia reginaFEBS JOURNAL, Issue 18 2006Kazuyo Fujikawa In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems. [source] Is my antibody-staining specific?EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008How to deal with pitfalls of immunohistochemistry Abstract Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto- and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false-positive results due to non-specific binding to tissue components or recognition of epitopes shared by several molecules. This ,Technical Spotlight' discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non-specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use. [source] Immunochemical and Immunohistochemical Identification of a Minor Collagen in Raw Muscles of Decapod MollusksJOURNAL OF FOOD SCIENCE, Issue 4 2000S. Mizuta ABSTRACT: Approximately 10% of total muscle collagen in 6 decapod molluskan species was recovered as minor collagenous fractions by limited pepsin digestion and differential salt precipitation. The main alpha components (,1) in these fractions showed similar peptide maps of V-8 protease and lysyl endopeptidase digests to each other and had reactivity to the antiserum against the al component of the minor collagen (named Type SQ-II) from Todarodes pacificus muscle in immunoblot analysis. These results suggested that the minor molecular species of collagen corresponding to Type SQ-II of Todarodes pacificus was widely distributed in the mantle muscle of decapod mollusks. In addition, immunohistochemical experiments revealed that Type SQ-II collagen distributed mainly in intramuscular thin connective tissue (endomysium), suggesting the functional importance of this molecule to the development of meat texture of decapod mollusks. [source] Cellular and subcellular localization of the GABAB receptor 1a/b subunit in the rat periaqueductal gray matterTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 5 2007Paolo Barbaresi Abstract The inhibitory effects of ,-aminobutyric acid (GABA)ergic neurotransmission in the periaqueductal gray matter (PAG) are mediated, at least partly, by metabotropic GABAB receptor subtypes whose cellular and subcellular localization is still unknown. We performed immunohistochemical experiments with an antibody against GABAB receptor subtype 1a/b (GABABR1a/b) by using light and electron microscopy. On light microscopy, GABABR1a/b immunoreactivity (IR) was in all columns, defined by cytochrome oxidase histochemistry. Neuropil labeling was strongest in the lateral portion of dorsolateral PAG. Labeled neurons, albeit not numerous, were in ventrolateral, dorsal, and medial subdivisions and were sparser in dorsolateral PAG. Labeling was mostly on the soma of PAG neurons. Sometimes GABABR1a/b IR spread along proximal dendrites; in these cases bipolar neurons were the most common type. On electron microscopy, GABABR1a/b IR was mainly on dendrites (54.92% of labeled elements) and axon terminals (21.90%) making synapses with labeled and unlabeled postsynaptic elements. Presynaptic labeling was also on unmyelinated and myelinated axons (overall 8% of all labeled elements). Postsynaptically, GABABR1a/b IR was at extrasynaptic sites on dendritic shafts; spines were always unlabeled. On axon terminals, GABABR1a/b IR was on extrasynaptic membranes and sometimes on presynaptic membrane specializations. Of the labeled elements, 13.03% elements were distal astrocytic processes (dAsPs) surrounding both symmetric and asymmetric synapses whose pre- and postsynaptic elements were often labeled. Immunoreactive dAsPs were around the soma and dendrites of both labeled and unlabeled neurons. These findings provide insights into the intrinsic PAG organization and suggest that presynaptic, postsynaptic, and glial GABAB receptors may play crucial roles in controlling PAG neuronal activity. J. Comp. Neurol. 505:478,492, 2007. © 2007 Wiley-Liss, Inc. [source] Identification of distinct gene expression profiles in the synovium of patients with systemic lupus erythematosusARTHRITIS & RHEUMATISM, Issue 5 2007A. Nzeusseu Toukap Objective Synovitis is a common feature of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), but the pattern of joint involvement differs in each disease. This study was undertaken to investigate the global gene expression profiles in synovial biopsy tissue from the swollen knees of untreated SLE patients (n = 6), RA patients (n = 7), and osteoarthritis (OA) patients (n = 6). Methods Synovial biopsy samples were obtained from the affected knees of patients in the 3 groups by needle arthroscopy. Half of the material was used for extraction of total RNA, amplification of complementary RNA, and high-density oligonucleotide spotted hybridization arrays. On the remaining tissue samples, real-time reverse transcription,polymerase chain reaction (RT-PCR) and immunohistochemical experiments were performed to confirm the microarray data. Results SLE synovial biopsy tissue displayed a significant down-regulation of genes involved in extracellular matrix (ECM) homeostasis and a significant up-regulation of interferon-inducible (IFI) genes. Real-time RT-PCR experiments confirmed the up-regulation of selected IFI genes (IFI27, IFI44, and IFI44L) in the SLE synovial tissue. Immunohistochemical analyses showed that 3 molecules involved in ECM regulation, chondroitin sulfate proteoglycan 2, latent transforming growth factor , binding protein 2, and fibroblast activation protein ,, were significantly down-regulated in SLE synovium. In contrast, immunostaining for IFI27, Toll-like receptor 4, and STAT-1 resulted in higher quantitative scores in SLE synovial tissue, which could be attributed to the fact that the RA samples had a large population of inflammatory cell infiltrates that were negative for these markers. Conclusion Arthritis in SLE has a very distinct molecular signature as compared with that in OA and RA, characterized by up-regulation of IFI genes and down-regulation of genes involved in ECM homeostasis. [source] | |||||||||||||