DOCSLIB.ORG

Immunohistochemical Staining Methods – Sixth Edition Immunohistochemical Staining Methods

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Immunohistochemical Staining Methods – Sixth Edition Immunohistochemical Staining Methods EDUCATIONAL IHC Guidebook Sixth Edition Education Guide Immunohistochemical Staining Methods Immunohistochemical Staining Methods – Sixth Edition Corporate Headquarters Australia Canada France Japan Poland United Kingdom Denmark +61 3 9357 0892 +1 905 335 3256 +33 1 64 53 61 44 +81 3 5802 7211 +48 58 661 1879 +44 (0)1 353 66 99 11 +45 44 85 95 00 Austria China Germany Korea Spain United States of America +43 1 408 43 34 0 +86 21 3612 7091 +49 40 69 69 470 +82 2 402 6775 +34 93 499 05 06 +1 805 566 6655 Belgium Denmark Ireland The Netherlands Sweden www.dako.com +32 (0) 16 38 72 20 +45 44 85 97 56 +353 1 479 0568 +31 20 42 11 100 +46 8 556 20 600 Represented in more Brazil Finland Italy Norway Switzerland than 100 countries +55 11 50708300 +358 9 348 73 950 +39 02 58 078 1 +47 23 14 05 40 +41 41 760 11 66 08002 07NOV13 Education Guide Immunohistochemical Staining Methods Sixth Edition Editors Clive R. Taylor, MD, D.Phil, Past Chair, Department of Pathology, University of Southern California; Past Council Member and President, Association of Pathology Chairs Lars Rudbeck, PhD, MSc, Scientific Editor, Dako Denmark Graphic Design Anja H. Sjørup, MSc, Graphic Designer, Dako Denmark © Copyright 2013 | Dako Denmark A/S, An Agilent Technologies Company. All rights reserved. No part of this book may be reproduced, copied or transmitted without written permission. Immunohistochemical Staining Methods | Table of Contents Table of Contents Immunohistochemical Staining Methods Part I: The Staining Process Preface.................................................................................................. 8 Chapter 1 | Introduction to Immunohistochemistry . 10 by C.R. Taylor 1.1 | Introduction.................................................................................. 11 1.2 | History of Immunohistochemistry . 13 1.3 | Standardization in Clinical Immunochemistry vs. Immunohistochemistry ..................15 1.4 | Growing Consensus for Standardization . 16 1.5 | Standardization Starts in the Surgery Room . 16 1.6 | Future Aspects for Standardization of Immunohistochemistry .....................................17 Chapter 2 | Fixation and Other Pre-Analytical Factors . 20 by E.C. Colley & R.H. Stead 2.1 | Introduction.................................................................................. 21 2.2 | Tissue Handling . 21 2.3 | Fixation . 23 2.4 | Tissue and Slide Processing . 25 2.5 | Special Tissue Preparations ..................................................... 26 2.6 | Control Material ........................................................................... 27 2.7 | Validation...................................................................................... 28 2.8 | Conclusions . 29 Chapter 3 | Antigen Retrieval . .30 by S-R Shi & C.R. Taylor 3.1 | Introduction.................................................................................. 31 3.2 | Major Factors that Influence the Effect of Antigen Retrieval .........................................32 3.3 | Standardization of AR – The “Test Battery” Approach . 34 3.4 | Application of AR Techniques – The Basic Principles . 36 3.5 | AR-IHC-based Research and Diagnostics . 36 3.6 | Reagents and Protocols ............................................................ 36 3.7 | Water Bath Methods................................................................... 36 3.8 | Pressure Cooker Heating .......................................................... 38 3.9 | Autoclave Heating ...................................................................... 39 3.10 | Microwave Oven Heating . 39 3.11 | Proteolytic Pre-treatment......................................................... 40 3.12 | Combined Proteolytic Pre-treatment and Heat-Induced Antigen Retrieval..............40 3.13 | Combined Deparaffinization and Antigen Retrieval . 41 3.14 | Conclusions............................................................................... 42 Table of Contents | Immunohistochemical Staining Methods Chapter 4 | Selection of the Primary Antibody . 46 by S. Nielsen 4.1 | Introduction.................................................................................. 47 4.2 | Seletion of the Proper Antibody . 47 4.3 | A New (Replacement) Antibody for an Old Marker . 49 4.4 | A New Antibody for New Marker in the Pathology Laboratory .....................................51 4.5 | A New Experiment in the Pathology Laboratory . 52 4.6 | Examples of Good and Poor Antibodies . 54 4.7 | Current and Future Challenges................................................ 57 Chapter 5 | Staining Protocol Optimization . 60 by L. Jacobsen, M. Nielsen, S. Månsson & L. Rudbeck 5.1 | Introduction.................................................................................. 61 5.2 | The Basis for an Optimal Staining Protocol . 62 5.3 | The protocol................................................................................. 64 5.4 | Reproducibility of IHC................................................................ 70 5.5 | Requirements for Controls . 70 5.6 | Verification and Validation of a Protocol . 73 5.7 | Guide to the Development of an Optimal Staining Protocol . 73 5.8 | Protocol Performance for Abnormal Tissue . 73 5.9 | Concluding remarks................................................................... 76 Chapter 6 | Immunohistochemistry Staining Methods . 78 by K. Petersen & H.C. Pedersen 6.1 | Introduction.................................................................................. 79 6.2 | Avidin-Biotin Immunohistochemistry . 79 6.3 | Polymer-Based Immunohistochemistry . 80 6.4 | Catalysed Signal Amplification (CSA) . 81 6.5 | Fluorescyl-tyramide Amplification . 82 6.6 | Improved Catalysed Signal Amplification (iCSA) . 82 6.7 | Multi-Staining Immunohistochemistry . ..
Load more

Recommended publications
  • Patterns of Green Fluorescent Protein Expression in Transgenic Plants
    Color profile: Generic CMYK printer profile Composite Default screen Plant Molecular Biology Reporter 18: 141a–141i, 2000 © 2000 International Society for Plant Molecular Biology. Printed in Canada. Publish by Abstract Patterns of Green Fluorescent Protein Expression in Transgenic Plants BRIAN K. HARPER1 and C. NEAL STEWART JR.2,* 1Novartis Agricultural Biotechnology Research, Inc., 3054 Cornwallis Rd., Research Triangle Park, NC 27709; 2Dept of Biology, University of North Carolina, Greensboro, NC 27402-6174 Abstract. Modified forms of genes encoding green fluorescent protein (GFP) can be mac- roscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP vi- sualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing trans- genic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice. Key words: expression patterns, green fluorescent protein, marker genes Patterns of IntroductionGFP expression Harper and Stewart Since the discovery of green fluorescent protein (GFP) from the jellyfish Aequorea victoria it has become a frequently used tool in biology.
    [Show full text]
  • Supplementary Materials and Method Immunostaining and Western Blot
    Supplementary Materials and Method Immunostaining and Western Blot Analysis For immunofluorescence staining, mouse and human cells were fixed with 4% paraformaldehyde- PBS for 15 min. Following Triton-X100 permeabilization and blocking, cells were incubated with primary antibodies overnight at 4°C following with Alexa 594-conjugated secondary antibodies at 4°C for 1 hour (Thermo Fisher Scientific, 1:1000). Samples were mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories) and immunofluorescence was detected using Olympus confocal microscopy. For western blot analysis, cells were lysed on ice using RIPA buffer supplemented with protease and phosphatase inhibitors (Sigma). Primary Antibodies for Immunostaining and Western Blot Analysis: Yap (14074, Cell Signaling), pYAP (4911, Cell Signaling), Lats1 (3477, Cell Signaling), pLats1( 8654, Cell Signaling), Wnt5a (2530, Cell Signaling), cleaved Caspase-3 (9661, Cell Signaling), Ki-67 (VP-K451, Vector Laboratories), Cyr61 (sc-13100, Santa Cruz Biotechnology), CTGF (sc-14939, Santa Cruz Biotechnology), AXL (8661, Cell Signaling), pErk (4376, Cell Signaling), pMEK (4376, Cell Signaling), Ck-19 (16858-1-AP, Proteintech), Actin (A2228, Sigma Aldrich), Vinculin (V4139, Sigma Aldrich), Kras (sc-30, Santa Cruz Biotechnology). Ectopic expression of YAP1 and WNT5A in mouse and human cells To generate YAP1S127A-expressing stable Pa04C cells, Pa04C cells were transfected with a linearized pcDNA3.1 plasmid with or without YAP1 cDNA containing S127A substitution. Two days post-transfection using Lipofectamine1000, cultures were selected in G418 (Sigma) and single clones were picked and expanded for further analysis. Overexpression of YAPS127A or WNT5A in human or mouse cells other than Pa04C were acheieved with lentivral infection. Briefly, lentivirus infection was performed by transfecting 293T cells with either GFP control, YAP1S127A, or WNT5A cloned in pHAGE lentivirus vector {EF1α promoter-GW-IRES-eGFP (GW: Gateway modified)}.
    [Show full text]
  • Mcb 407-Immunology and Immunochemistry-Lecture Note
    MCB 407-IMMUNOLOGY AND IMMUNOCHEMISTRY-LECTURE NOTE DR. D. A. OJO BRIEF HISTORICAL REVIEW OF IMMUNOLOGY The mechanism by which antibody are formed has been debated for years. It was proposed that the specificity of an antibody molecule was determined both by its amino acid sequence but by the molding of the peptide chain around the antigenic determinant. This theory lost favour when it became apparent that antibody-forming cells were devoid of antigen and that antibody specificity was a function of amino acid sequence. At present, the CLONAL (proposed by Burnete) SELECTION THEORY is widely accepted. It holds that an immunologically responsive cell can respond to only one antigen or a closely related group of antigens and that this property is inherent in the cell before the antigen is encountered. According to the clonal selection theory, each individual is endowed with a very large pool of lymphocytes, each of which is capable of responding to a different antigen. When the antigen enters the body, it selects the lymphocyte which has the best “fit” by virtue of a surface receptor. The antigen binds to this antibody-like receptor, and the cell is stimulated to proliferate and form a clone of cells. Thus, selected cells quickly differentiate into plasma cells and secrete antibody which is specific for the antigen which served as the original selecting agent (or a closely related group of antigens). The History of Blood Transfusion Man’s centuries-long desire to perform blood transfusion as a therapeutic procedure forms the cornerstone of the modern science of immunohematology. At present time, the use of whole blood is a well-accepted and commonly employed measure without which many modern surgical procedures could not be carried out.
    [Show full text]
  • CCR7 Antibody (Y59) NB110-55680
    Product Datasheet CCR7 Antibody (Y59) NB110-55680 Unit Size: 0.1 ml Store at -20C. Avoid freeze-thaw cycles. Publications: 9 Protocols, Publications, Related Products, Reviews, Research Tools and Images at: www.novusbio.com/NB110-55680 Updated 5/28/2019 v.20.1 Earn rewards for product reviews and publications. Submit a publication at www.novusbio.com/publications Submit a review at www.novusbio.com/reviews/destination/NB110-55680 Page 1 of 6 v.20.1 Updated 5/28/2019 NB110-55680 CCR7 Antibody (Y59) Product Information Unit Size 0.1 ml Concentration This product is unpurified. The exact concentration of antibody is not quantifiable. Storage Store at -20C. Avoid freeze-thaw cycles. Clonality Monoclonal Clone Y59 Preservative 0.01% Sodium Azide Isotype IgG Purity Tissue culture supernatant Buffer 49% PBS, 0.05% BSA and 50% Glycerol Target Molecular Weight 45 kDa Product Description Host Rabbit Gene ID 1236 Gene Symbol CCR7 Species Human, Mouse, Rat, Monkey Reactivity Notes May cross reacts with Rhesus monkey. Specificity/Sensitivity The antibody does not cross-react with other G-protein coupled receptor 1 family members. Immunogen A synthetic peptide corresponding to residues in N-terminal extracellular domain of human CKR7 was used as immunogen. Notes Produced using Abcam's RabMab® technology. RabMab® technology is covered by the following U.S. Patents, No. 5,675,063 and/or 7,429,487. Product Application Details Applications Western Blot, Immunocytochemistry/Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Frozen, Immunohistochemistry- Paraffin, Immunoprecipitation, Flow Cytometry (Negative) Recommended Dilutions Western Blot 1:1000-10000, Immunohistochemistry 1:10-1:500, Immunocytochemistry/Immunofluorescence 1:250, Immunoprecipitation 1:10, Immunohistochemistry-Paraffin 1:250, Immunohistochemistry-Frozen 1:250, Flow Cytometry (Negative) Application Notes This product is useful for: Western Blot, Immunohistochemistry-Paraffin, Immunocytochemistry, Immunoprecipitation.
    [Show full text]
  • TITAN IV Immunoelectrophoresis Procedure
    TITAN IV Immunoelectrophoresis Procedure Cat. No. 9050 and 9061 Helena Laboratories Titan IV Immunoelectrophoresis (IEP) is intended for barbital-sodium barbital buffer with 0.1% sodium azide added semiquantitative determinations by immunoelectrophoresis. as a preservative. WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. This SUMMARY product contains sodium azide. Refer to the sodium azide Immunoelectrophoresis (IEP) combines two techniques, warning. electrophoresis and immunodiffusion. In this two-part procedure, Preparation for Use: The plates are ready for use as proteins in a serum or urine sample are first separated packaged. according to charge by electrophoresis. Then, antisera Storage and Stability: Plates should be stored flat, in the complimentary to the proteins under study are applied to the protective packaging, at 2°C to 8°C and are stable until the plate and allowed to diffuse. When a favorable antigen to expiration date indicated on the label. DO NOT FREEZE antibody ratio exists, a precipitin arc will form on the plate. PLATES OR EXPOSE THEM TO EXCESSIVE HEAT. IEP is used for the diagnosis and differential diagnosis of Signs of Deterioration: The plates should have a smooth, monoclonal gammopathies when using serum or urine clear agarose surface. Discard the plates if they appear 1-4 specimens. The method is also used for a number of other cloudy, show bacterial growth, or if they have been exposed to purposes, including screening for circulating immune freezing (a cracked or bubbled surface) or excessive heat (a complexes, characterization of cryoglobulinemia and dried, thin surface). pyroglobulinemia, recognition and characterization of antibody 2. Antisera for Assay of Immunoglobulins syndromes, and recognition and characterization of the various Antiserum to Human IgG, Cat.
    [Show full text]
  • IMMUNOELECTROPHORESIS TEST Dr
    [Type text] IMMUNOELECTROPHORESIS TEST Dr. Daljeet Chhabra Department of Veterinary Microbiology College of Veterinary Science and A.H., Mhow NDVSU, Jabalpur The term “immunoelectrophoresis” was first coined by Grabar and Williams in 1953. Immunoelectrophoresis refers to precipitation in agar under an electric field. It is a process of a combination of immuno-diffusion and electrophoresis. An antigen mixture is first separated into its component parts by electrophoresis and then tested by double immuno-diffusion. Antigens are placed into wells cut in a gel (without antibody) and electrophoresed. A trough is then cut in the gel into which antibodies are placed. The antibodies diffuse to meet diffusing antigens, and lattice formation and precipitation occur permitting determination of the nature of the antigens. Principle: When an electric current is applied to a slide layered with gel, the antigen mixture placed in wells is separated into individual antigen components according to their charge and size. Following electrophoresis, the separated antigens are reacted with specific antisera placed in troughs parallel to the electrophoretic migration and diffusion is allowed to occur. Antiserum present in the trough moves toward the antigen components resulting in the formation of separate precipitin lines in 18-24 hrs, each indicating reaction between individual proteins with its antibody. Materials required: Agarose gel, slides, well cutters, Immunoelectrophoresis machine, buffer, etc. Procedure: 1. Agarose gel is prepared on a glass slide put in a horizontal position. 2. After well cutting, sample is added in well. 3. The gel is placed into the electrophoresis chamber with the samples on the cathodic side, and electrophoresis runs for 20 mins/ 100 volts.
    [Show full text]
  • Memoranaurns by the Participants in Signes Par Les Partici- I the Meeting
    Memoranda are state- Les Memorandums ments concerning the exposent les conclu- /e , conclusions or recom- sions et recomman- M e mmooranrantedaa mendations of certain dations de certaines / a t w / /WHO scientific meet- /reunions scientifiques ings; they are signed de /'OMS; ils sont Memoranaurns by the participants in signes par les partici- I the meeting. pants a ces reunions. Bulletin ofthe World Health Organization, 62 (2): 217-227 (1984) © World Health Organization 1984 Immunodiagnosis simplified: Memorandum from a WHO Meeting* Technologies suitable for the development ofsimplified immunodiagnostic tests were reviewed by a Working Group of the WHO Advisory Committee on Medical Research in Geneva in June 1983. They included agglutination tests and use ofartificialparticles coated with immunoglobulins, direct visual detection of antigen-antibody reactions, enzyme- immunoassays, and immunofluorescence and fluoroimmunoassays. The use of mono- clonal antibodies,in immunodiagnosis and of DNA /RNA probes to identify viruses was also discussed in detail. The needfor applicability of these tests at three levels, i.e., field conditions (or primary health care level), local laboratories, and central laboratories, was discussed and their use at thefield level was emphasized. Classical serological techniques have been used for All these tests can be carried out in laboratories that a long time for diagnostic purposes, e.g., for con- are equipped with basic instruments as well as special- firmation of clinical diagnoses, epidemiological ized apparatus (e.g., gamma counters for RIA, ultra- studies, testing of blood donors, etc. Some of these violet microscopes for IMF, etc.), which are usually techniques have been standardized to a high degree of available only in the larger, central laboratories.
    [Show full text]
  • Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 E-Mail: [email protected]
    PharMingen International United States Canada PharMingen Europe Japan PharMingen Canada Tel 619-812-8800 Asia Pacific Becton Dickinson GmbH Nippon Becton Dickinson Toll-Free 1-888-259-0187 Orders 1-800-848-6227 BD Singapore HQ PharMingen Europe Company Ltd. Tel 905-542-8028 Tech Service 1-800-825-5832 Tel (65) 860-1478 Tel (49) 40 532 84 48 0 Tel (81) 3 541-382-51 Fax 905-542-9391 Fax 619-812-8888 Cytokine Immunocytochemistry Fax (65) 860-1590 Fax (49) 40 531 58 92 Fax (81) 3 541-381-55 e-mail: [email protected] http://www.pharmingen.com Africa Germany Latin/South America Spain Becton Dickinson Worldwide Inc. Becton Dickinson GmbH BDIS (USA) Becton Dickinson S.A. Reagents and Techniques Tel (254) 2 449 608 HQ PharMingen Europe Tel (408) 954-2157 Tel (34) 91 848 8100 Fax (254) 2 449 619 Tel (49) 40 532 84 48 0 Fax (408) 526-1804 Fax (34) 91 848 8105 Fax (49) 40 531 58 92 Orders for Microscopic Analysis of Cytokine-Producing Cells Australia Malaysia Tel (34) 91 848 8182 Becton Dickinson Pty Ltd Greece Becton Dickinson Sdn Bhd Fax (34) 91 848 8104 Tel (612) 9978-6800 Becton Dickinson Hellas S.A. Tel (03) 7571323 Fax (612) 9978-6850 Tel (30) 1 9407741 Fax (03) 7571153 Sweden Fax (30) 1 9407740 Becton Dickinson AB Austria Mexico Tel (46) 8 775 51 00 Becton Dickinson GmbH Hong Kong Becton Dickinson de Mexico Fax (46) 8 645 08 08 HQ PharMingen Europe Becton Dickinson Asia Ltd Tel (52-5) 237-12-98 Tel (49) 40 532 84 48 0 Tel (852) 2572-8668 Fax (52-5) 237-12-93 Switzerland Fax (49) 40 531 58 92 Fax (852) 2520-1837 Becton Dickinson GmbH Middle East HQ PharMingen Europe Belgium Hungary Becton Dickinson Tel (41) 06 1-385 4422 Becton Dickinson Benelux N.V.
    [Show full text]
  • Scholarworks@UNO
    University of New Orleans ScholarWorks@UNO University of New Orleans Theses and Dissertations Dissertations and Theses Summer 8-4-2011 Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria Avijit Biswas University of New Orleans, [email protected] Follow this and additional works at: https://scholarworks.uno.edu/td Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Biswas, Avijit, "Identification and characterization of enzymes involved in the biosynthesis of different phycobiliproteins in cyanobacteria" (2011). University of New Orleans Theses and Dissertations. 446. https://scholarworks.uno.edu/td/446 This Dissertation-Restricted is protected by copyright and/or related rights. It has been brought to you by ScholarWorks@UNO with permission from the rights-holder(s). You are free to use this Dissertation-Restricted in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/or on the work itself. This Dissertation-Restricted has been accepted for inclusion in University of New Orleans Theses and Dissertations by an authorized administrator of ScholarWorks@UNO. For more information, please contact [email protected]. Identification and characterization of enzymes involved in biosynthesis of different phycobiliproteins in cyanobacteria A Thesis Submitted to the Graduate Faculty of the University of New Orleans in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Chemistry (Biochemistry) By Avijit Biswas B.S.
    [Show full text]
  • Ultrastructural Localization of Glial Fibrillary Acidic Protein in Mouse Cerebellum by Immunoperoxidase Labeling
    Ultrastructural localization of glial fibrillary acidic protein in mouse cerebellum by immunoperoxidase labeling The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Schachner, M. 1977. “Ultrastructural Localization of Glial Fibrillary Acidic Protein in Mouse Cerebellum by Immunoperoxidase Labeling.” The Journal of Cell Biology 75 (1) (October 1): 67–73. doi:10.1083/jcb.75.1.67. Published Version 10.1083/jcb.75.1.67 Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:32705572 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ULTRASTRUCTURAL LOCALIZATION OF GLIAL FIBRILLARY ACIDIC PROTEIN IN MOUSE CEREBELLUM BY IMMUNOPEROXIDASE LABELING M. SCHACHNER, E. T. HEDLEY-WHYTE, D. W. HSU, G. SCHOONMAKER, and A. BIGNAMI From the Department of Neuroiogy-Neuropathology, Harvard Medical School and the Departments of Neuroscience and Pathology (Neuropathology) and the Mental Retardation Research Center, Children's Hospital Medical Center, Boston, Massachusetts 02115. Dr. Bignami's present address is Spinal Cord Injury Research, West Roxbury V. A. Hospital, West Roxbury, Massachusetts 02132. ABSTRACT Glial fibrillary acidic protein was localized at the electron microscope level in the cerebellum of adult mice by indirect immunoperoxidase histology. In confirma- tion of previous studies at the light microscope level, the antigen was detectable in astrocytes and their processes, but not in neurons or their processes, or in oligodendroglia.
    [Show full text]
  • Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology
    International Journal of Molecular Sciences Review Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology Jean-Denis Pedelacq 1,* and Stéphanie Cabantous 2,* 1 Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, 31077 Toulouse, France 2 Centre de Recherche en Cancérologie de Toulouse (CRCT), Inserm, Université Paul Sabatier-Toulouse III, CNRS, 31037 Toulouse, France * Correspondence: [email protected] (J.-D.P.); [email protected] (S.C.) Received: 15 June 2019; Accepted: 8 July 2019; Published: 15 July 2019 Abstract: Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite » split-FP,that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein–protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology. Keywords: fluorescent protein; superfolder; split-GFP; bipartite; tripartite; folding; PPI 1. Superfolder Fluorescent Proteins: Progenitor of Split Fluorescent Protein (FP) Systems Previously described mutations that improve the physical properties and expression of green fluorescent protein (GFP) color variants in the host organism have already been the subject of several reviews [1–4] and will not be described here.
    [Show full text]
  • Diagnostic Potential of the Haemagglutination
    Bulgarian Journal of Veterinary Medicine (2007), 10 , N o 3, 169 −178 DIAGNOSTIC POTENTIAL OF THE HAEMAGGLUTINATION INHIBITION TEST, THE IMMUNODIFFUSION TEST AND ELISA FOR DETECTION OF ANTIBODIES IN CHICKENS, INTRAVENOUSLY INFECTED WITH A/DUCK/BULGARIA/05 H6N2 AVIAN INFLUENZA VIRUS ISOLATE I. S. ZARKOV Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria Summary Zarkov, I., 2007. Diagnostic potential of the haemagglutination inhibition test, the immuno- diffusion test and ELISA for detection of antibodies in chickens, intravenously infected with A/duck/Bulgaria/05 H6N2 avian influenza virus isolate. Bulg. J. Vet. Med. , 10 , No 3, 169 −178. After experimental infection of chickens with an avian influenza viral isolate A/duck/Bulgaria/05 H6N2, the potential of the haemagglutination inhibition test (HIT), the immunodiffusion test (IDT) and enzyme-linked immunosorbent assay (ELISA) for antibody detection was evaluated. The results evidenced that in birds, haemagglutinins, precipitins and IgG antibodies, detectable with ELISA, were formed. The percentage of chickens with subtype-specific antibodies was the highest by the 21 st day of infection (100 %), followed by the 14 th (66.7 %) and the 28 th (55.6 %) days, and was the lowest by the 7 th day (44.4 %). Serum titres ranged between 1:4 and 1:256 with predomination of 1:8 and 1:16 titres (29.2 % each). The mean arithmetic titre for the experiment was 1:38.2. The highest percentage of chickens with precipitins was observed by the 14th day (55.6 %) followed by the 7 th , 21 st and the 28 th days with 33.3 % each.
    [Show full text]