Bı∙Olegend 2012–2013
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Supplement Table of Contents
Supplement Table of Contents: Materials and Methods Supplement. Supplement Figure 1. IL-34 and CSF-1 are expressed with advancing lupus nephritis in MRL-Faslpr mice Supplement Figure 2. Submandibular inflammation (sialadenitis) is suppressed in IL-34 KO MRL- Faslpr mice. Supplement Figure 3. Mø, T and B cell accumulation is suppressed in IL-34 KO MRL- Faslpr mice. Supplement Figure 4. CSF-1 does not compensate for the absence of IL-34 in the kidney of IL-34 KO MRL- Faslpr mice. Supplement Figure 5. Mø skew towards cyto-destructive M1 phenotype during lupus nephritis in MRL- Faslpr mice and in BM Mø co-cultured with hypoxic TEC. Supplement Figure 6. Proliferating B cells are suppressed in IL-34 KO MRL-Faslpr mice with lupus nephritis. Supplement Figure 7. IL-34 correlates with Mø and T cells and histopathology disease activity in type II-IV lupus nephritis Supplement Table 1. Study cohort demographic and clinical characteristics. Supplement Table 2. Antibodies used for immunostaining. Supplement Table 3. qPCR primers used to detect mRNAs. Supplement Table 4. Antibodies used for FACS. Supplement Materials and Methods Skin Lesions We scored the skin lesions by gross pathology using a grade of 0–3 (0 = none; 1 = mild (snout and ears); 2 = moderate, < 2 cm (snout, ears, and intrascapular); 3 = severe, 2-4 cm (snout, ears, and intrascapular); and 4 = very severe, >4 cm (snout, ears, and intrascapular). Moreover, we assessed the incidence of skin lesions that showed grade 2 or more. β-Galactosidase Kidneys were fixed in 4% paraformaldehyde for 3 hours at 4°C, embedded and sectioned at 20-µm thick sections. -
Cellular and Structural Neuroscience Markers SMI® Monoclonals and Β-Tubulin Antibodies
Cellular and Structural Neuroscience Markers SMI® Monoclonals and β-Tubulin Antibodies BioLegend is proud to offer SMI® monoclonal antibodies in our class of neuroscience antibody research tools. Originally developed by Dr. Ludwig Sternberger, these antibodies have become the gold standard as neural and glial markers and have an extensive publication history in scientific literature. In addition to our β-Tubulin antibodies, the SMI® product line offers researchers high quality reagents to support investigations into neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease. Key Features: Rat hippocampus stained with anti-GFAP (red) and anti-NF-H (822601 green). Photo courtesy of Molecular Expressions. • Antibodies specific for phosphorylated and non- phosphorlylated neurofilaments, myelin basic proteins, and GFAP. • Excellent selection of specificities for oligodendrocytes, astrocytes, and other glial cells. • Each high quality antibody is quality tested for specific, robust, and consistent performance. • Experienced technical support staff and in-house generated evaluation data. Rat cortical neurons and glia in mixed tissue culture stained with anti- NF-H (red) and GFAP (green, 840001). The nuclei are stained with Hoescht dye (blue). Photo courtesy of EnCor Biotechnology Inc. To explore the full line of products, visit: Human brain section stained with anti-GFAP (brown, 837201), counterstained with Hematoxylin (blue). biolegend.com/neuroscience_products Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.5800 biolegend.com 07-0092-01 World-Class Quality | Superior Customer Support | Outstanding Value Cellular and Structural Neuroscience Markers Phosphorylated Neurofilaments Non-Phosphorylated Neurofilaments Specificity Clone Reactivity Application(s) Cat. No. Specificity Clone Reactivity Application(s) Cat. No. -
TNF Increases Tyrosine Hydroxylase Expression in Human Monocytes
bioRxiv preprint doi: https://doi.org/10.1101/2021.04.13.439627; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. TNF increases Tyrosine Hydroxylase expression in human monocytes Madison Francisa1, Martin Badova1, Gerry Shaw1,2, Anthony Collins1, Douglas R. Miller1, Carissa A. Hansen1, Phillip Mackie1, Malú Gámez Tansey1, Abeer Dagra1, Irina Madorsky2, Adolfo Ramirez-Zamora3, Michael S. Okun1,3, Wolfgang J. Streit1, Adithya Gopinath*1, Habibeh Khoshbouei1 1-University of Florida, Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, Norman Fixel Institute for Neurological Diseases 2-Encor Biotechnology Inc. 4949 SW 41st Blvd Suite 40, Gainesville FL 32608 3-University of Florida, Department of Neurology a equal contribution Corresponding Author: *Adithya Gopinath [email protected] Number of Pages (incl. References): 28 Number of Tables: 4 Number of Figures: 6 Number of References: 72 Abbreviations: No non-standard abbreviations used 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.04.13.439627; this version posted April 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. -
Neuroscience Research Products
Neuroscience Research Products BioLegend is ISO 9001:2008 and ISO 13485:2003 Certified Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.5800 biolegend.com 02-0010-00 World-Class Quality | Superior Customer Support | Outstanding Value Unlocking the biology of the brain is a research With the acquisition of the Covance antibody product imperative that has garnered much attention from portfolio, BioLegend signaled its commitment to public and private entities in recent years. From the enabling legendary discovery within the neuroscience National Football League to the U.S. Department research community. BioLegend is making further of Defense, traumatic brain injury is being heavily investments in the portfolio, becoming a leading investigated. In addition, the BRAIN Initiative and provider of reagents known for quality and innovation, EU Initiative (humanbrainproject.eu) are focused on and helping scientists advance the understanding of innovative research and technology that increases our brain biology, interrogate their pathways, and identify understanding of the brain. Neurodegenerative diseases potential biomarkers that may improve the success already constitute a public health and economic of clinical programs for neurodegenerative diseases. crisis, and as the population ages, the urgent need for better treatments for these diseases is intensified. Protein Aggregation Protein aggregation is a hallmark of neurodegenerative disease pathology (e.g. beta-amyloid plaques in Alzheimer's disease, Lewy bodies in Parkinson's disease, etc). Post-translational modification of key proteins is known to be a critical factor in the pathways that regulate formation, deposition and propagation of these aggregates. BioLegend offers a portfolio of products that set the standard for quality, allowing researchers to detect native and modified forms of key proteins in a wide range of applications. -
Product Data Sheet Anti-GFAP
Version: 1 Revision Date: 2015-03-27 Product Data Sheet Anti-GFAP Catalog # / Size: 840001 / 100 µl Previously: Covance Catalog# PRB-571C Clone: Poly28400 Isotype: Rabbit Polyclonal Immunogen: This antiserum was made with a preparation of recombinant GFAP expressed in bacteria and highly purified. Subsequent boosts were performed with purified GFAP from bovine spinal cord. Reactivity: Human, Mouse, Rat, Cat Preparation: Serum Concentration: Lot-specific (please contact technical support for concentration and total µg amount) Rat cortical neurons and glia in mixed tissue culture stained with NF-H (red) Storage: Store at -20°C. Upon initial thawing, apportion into working aliquots and and GFAP (green). The nuclei are store at -20°C. Avoid repeated freeze-thaw cycles to prevent denaturing stained with Hoescht dye (blue). the antibody. For long-term storage, keep the antibody at -80°C. Photo courtesy of EnCor Biotechnology Inc. Applications: Applications: WB, IF, ICC Recommended Usage: Each lot of this antibody is quality control tested by Western blotting. The optimal working dilution should be determined for each specific assay condition. • WB: 1:10,000* • IF: 1:1,000 (when using fluorescent secondary ab) or 1:5,000 (when using peroxidase labeled secondary ab) Application Notes: This antiserum is effective in immunoblotting (WB), immunofluorescence (IF) and immunocytochemistry (ICC). * Predicted MW = 55 kD & may see one at 48 kD (a breakdown product) Application References: 1. Brenner M et al. Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease. Nat. Genet. 27:117-120, Rat hippocampus stained with rabbit 2001. anti-GFAP, (red, AF568) and chicken 2. -
Reporting Summary R E P
n a t u r e Corresponding author(s): NCOMMS-19-10152A r e s e Last updated by author(s): Feb 21, 2020 a r c h | Reporting Summary r e p Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency o r t i in reporting. For further information on Nature Research policies, seeAuthors & Referees and theEditorial Policy Checklist . n g s u m m a r Statistics y For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section. n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable. -
Cell Structure & Function
Cell Structure & Function Antibodies and Reagents BioLegend is ISO 13485:2003 Certified Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.5800 biolegend.com 02-0012-02 World-Class Quality | Superior Customer Support | Outstanding Value Table of Contents Introduction ....................................................................................................................................................................................3 Cell Biology Antibody Validation .............................................................................................................................................4 Cell Structure/ Organelles ..........................................................................................................................................................8 Cell Development and Differentiation ................................................................................................................................10 Growth Factors and Receptors ...............................................................................................................................................12 Cell Proliferation, Growth, and Viability...............................................................................................................................14 Cell Cycle ........................................................................................................................................................................................16 Cell Signaling ................................................................................................................................................................................18 -
Tnfα Increases Tyrosine Hydroxylase Expression in Human Monocytes
www.nature.com/npjparkd ARTICLE OPEN TNFα increases tyrosine hydroxylase expression in human monocytes ✉ Adithya Gopinath 1,4 , Martin Badov1,4, Madison Francis1,4, Gerry Shaw1,2, Anthony Collins1, Douglas R. Miller 1, Carissa A. Hansen1, Phillip Mackie1, Malú Gámez Tansey1, Abeer Dagra1, Irina Madorsky2, Adolfo Ramirez-Zamora3, Michael S. Okun 1,3, Wolfgang J. Streit1 and Habibeh Khoshbouei 1 Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinson’s disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients’ monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNFα), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNFα in PD. -
Epitope Tag Antibodies & Affinity Gels
Epitope Tag Antibodies for Epitope Tag Antibodies Flow Cytometry Epitope tag antibodies are not only for western blotting and & Affinity Gels immunoprecipitation! These antibodies can also complement your flow cytometry workflow by facilitating the identification of cells For Purification & Research Applications based on the expression of your transfected protein. BioLegend offers antibodies to HA, DYKDDDDK, His, and GFP epitope tags for flow cytometry applications. For a comprehensive list of available Easy Purification formats, refer to the table on the reverse side. Tagging proteins of interest expands detectability of recombinant HA-Tag Transfected Cells Identified by Intracellular Flow Cytometry proteins. By incorporating an epitope tag into the sequence of a recombinant protein, antibodies can be used for surveillance of the tagged fusion protein. BioLegend has a wide range of antibodies and affinity gels for many epitope tags, including Myc, His, DYKDDDDK, GST, ell Number GFP, and HA. The epitope tag antibodies are extremely specific and provide outstanding performance for protein purification, western blot, flow cytometry, and immunoprecipitation. For Relative C your convenience, many of our epitope tag antibodies can be purchased immobilized on agarose beads as an affinity matrix 0 103 104 105 or affinity gel for simple purification. Our affinity gels have been Log Fluorescence Intensity tested to be reusable at least five times. CHO-K1 cells (open histogram) or HA tag stably transfected cells (filled histogram) were fixed with Fixation Buffer, permeabilized with True-Phos™ Perm Buffer (Cat. No. 425401), then intracellularly Stable Activity of anti-c-Myc Agarose Over Ten Rounds of stained with HA.11 Epitope Tag (Clone 16B12) PE (Cat. -
Microscopy Reagents for Immunocytochemistry and Immunohistochemistry
Microscopy Reagents for Immunocytochemistry and Immunohistochemistry BioLegend is ISO 13485:2016 Certified Toll-Free Tel: (US & Canada): 1.877.BIOLEGEND (246.5343) Tel: 858.768.5800 biolegend.com 02-0011-02 World-Class Quality | Superior Customer Support | Outstanding Value Immunohistochemistry and Immunofluorescence: What’s in a Name? Imaging platforms enable understanding of subcellular localization, function, activity and health in a single cell culture or a network of cells comprising a tissue. For each biological question that could benefit from quantitative, structural, spatially relevant information, there is a sophisticated microscopy platform that is best designed for the application at hand. Based on the biological application and the microscope of choice, there is an array of both chemical and antibody-based reagents to enable visualization. The biological application, the instrument and the reagents must all be well matched for ultimate success. Immunocytochemistry (ICC) is often called immunofluorescence (IF) and is characterized by imaging primary cells or cell lines in culture. Immunohistochemistry (IHC) is simply the detection of antibodies in tissue sections, whether it be by chromogenic or fluorescent realization methods. IHC-P indicates the antibody is useful in formalin-fixed paraffin-embedded (FFPE) sections and IHC-F indicates the antibody is only useful in tissue that has been fixed and frozen prior to sectioning. If there is only an IHC designation, check with the literature citations or additional information provided by the manufacturer to determine the method of tissue preparation compatible with each reagent. Widefield vs. Confocal Microscopy Widefield microscopy is the most common and accessible imaging platform. Historically, widefield microscopes relied on a mercury arc lamp as the primary excitation source, and excitation and emission filters to choose specific wavelengths of light to be matched to the reagent combination.