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  • 1. Al-Khalili Szigyarto, Cristina
    et al.
    Sibbons, P.
    Williams, G.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Metcalfe, S. M.
    The E3 Ligase Axotrophin/MARCH-7: Protein Expression Profiling of Human Tissues Reveals Links to Adult Stem Cells2010In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 58, no 4, p. 301-308Article in journal (Refereed)
    Abstract [en]

    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)

  • 2. Andersson, Ann-Catrin
    et al.
    Stromberg, Sara
    Backvall, Helena
    Kampf, Caroline
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Wester, Kenneth
    Ponten, Fredrik
    Analysis of protein expression in cell microarrays: A tool for antibody-based proteomics2006In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 54, no 12, p. 1413-1423Article in journal (Refereed)
    Abstract [en]

    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.

  • 3. Andersson, Sandra
    et al.
    Konrad, Anna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ashok, Nikhil
    Pontén, Fredrik
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Protein Technology.
    Asplund, Anna
    Antibodies Biotinylated Using a Synthetic Z-domain from Protein A Provide Stringent In Situ Protein Detection2013In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 61, no 11, p. 773-784Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Brismar, Hjalmar
    et al.
    KTH, Superseded Departments, Physics.
    Trepte, O
    Ulfhake, B
    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.1995In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 43, no 7Article in journal (Refereed)
    Abstract [en]

    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.

  • 5. Edqvist, Per-Henrik D.
    et al.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallström, Bjorn M.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Danielsson, Angelika
    Edlund, Karolina
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Pontén, Fredrik
    Expression of Human Skin-Specific Genes Defined by Transcriptomics and Antibody-Based Profiling2015In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 63, no 2, p. 129-141Article in journal (Refereed)
    Abstract [en]

    To increase our understanding of skin, it is important to define the molecular constituents of the cell types and epidermal layers that signify normal skin. We have combined a genome-wide transcriptomics analysis, using deep sequencing of mRNA from skin biopsies, with immunohistochemistry-based protein profiling to characterize the landscape of gene and protein expression in normal human skin. The transcriptomics and protein expression data of skin were compared to 26 (RNA) and 44 (protein) other normal tissue types. All 20,050 putative protein-coding genes were classified into categories based on patterns of expression. We found that 417 genes showed elevated expression in skin, with 106 genes expressed at least five-fold higher than that in other tissues. The 106 genes categorized as skin enriched encoded for well-known proteins involved in epidermal differentiation and proteins with unknown functions and expression patterns in skin, including the C1orf68 protein, which showed the highest relative enrichment in skin. In conclusion, we have applied a genome-wide analysis to identify the human skin-specific proteome and map the precise localization of the corresponding proteins in different compartments of the skin, to facilitate further functional studies to explore the molecular repertoire of normal skin and to identify biomarkers related to various skin diseases.

  • 6. Gardberg, Maria
    et al.
    Heuser, Vanina D.
    Iljin, Kristiina
    Kampf, Caroline
    Uhlen, Mathias
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Carpen, Olli
    Characterization of Leukocyte Formin FMNL1 Expression in Human Tissues2014In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 62, no 6, p. 460-470Article in journal (Refereed)
    Abstract [en]

    Formins are cytoskeleton regulating proteins characterized by a common FH2 structural domain. As key players in the assembly of actin filaments, formins direct dynamic cytoskeletal processes that influence cell shape, movement and adhesion. The large number of formin genes, fifteen in the human, suggests distinct tasks and expression patterns for individual family members, in addition to overlapping functions. Several formins have been associated with invasive cell properties in experimental models, linking them to cancer biology. One example is FMNL1, which is considered to be a leukocyte formin and is known to be overexpressed in lymphomas. Studies on FMNL1 and many other formins have been hampered by a lack of research tools, especially antibodies suitable for staining paraffin-embedded formalin-fixed tissues. Here we characterize, using bioinformatics tools and a validated antibody, the expression pattern of FMNL1 in human tissues and study its subcellular distribution. Our results indicate that FMNL1 expression is not restricted to hematopoietic tissues and that neoexpression of FMNL1 can be seen in epithelial cancer.

  • 7. Paavilainen, Linda
    et al.
    Edvinsson, Åsa
    Asplund, Anna
    Hober, Sophia
    KTH, School of Biotechnology (BIO), Proteomics.
    Kampf, Caroline
    Pontén, Fredrik
    Wester, Kenneth
    The Impact of Tissue Fixatives on Morphology and Antibody-based Protein Profiling in Tissues and Cells2010In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 58, no 3, p. 237-246Article in journal (Refereed)
    Abstract [en]

    Pathology archives harbor large amounts of formalin-fixed, paraffin-embedded tissue samples, used mainly in clinical diagnostics but also for research purposes. Introduction of heat-induced antigen retrieval has enabled the use of tissue samples for extensive immunohistochemical analysis, despite the fact that antigen retrieval may not recover all epitopes, owing to alterations of the native protein structure induced by formalin. The aim of this study was to investigate how different fixatives influence protein recognition by immunodetection methods in tissues, cell preparations, and protein lysates, as compared with formalin. Seventy-two affinity-purified polyclonal antibodies were used to evaluate seven different fixatives. The aldehyde-based fixative Glyo-fixx proved to be excellent for preservation of proteins in tissue detected by immunohistochemistry (IHC), similar to formalin. A non-aldehyde-based fixative, NEO-FIX was superior for fixation of cultured cells, in regard to morphology, and thereby also advantageous for IHC. Large variability in the amount of protein extracted from the differently fixed tissues was observed, and the HOPE fixative provided the overall highest yield of protein. In conclusion, morphological resolution and immunoreactivity were superior in tissues fixed with aldehyde-based fixatives, whereas the use of non-aldehyde-based fixatives can be advantageous in obtaining high protein yield for Western blot analysis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 58:237-246, 2010)

  • 8.
    Treviño-Villareal, J Humberto
    et al.
    Dept of Environmental Health, Harvard School of Public Health.
    Cotanche, Douglas A
    Dept of Environmental Health, Harvard School of Public Health.
    Sepúlveda, Rosalinda
    Dept of Environmental Health, Harvard School of Public Health.
    Bortoni, Magda E
    Dept of Environmental Health, Harvard School of Public Health.
    Manneberg, Otto
    Dept of Environmental Health, Harvard School of Public Health.
    Udagawa, Taturo
    Vertex Pharmaceuticals, Cambridge, Massachusetts.
    Rogers, Rick A
    Dept of Environmental Health, Harvard School of Public Health.
    Host-derived pericytes and Sca-1+ cells predominate in the MART-1 stroma fraction of experimentally induced melanoma2011In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 59, p. 1060-1075Article in journal (Refereed)
    Abstract [en]

    Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti-MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1-, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31-, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.

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