Chapter Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence micro scopy Fluorescent immunocytochemistry Fluorescent immunohistochem istry Indirect immunocytochemistry Immunostain - PDF document

Chapter  Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence micro scopy Fluorescent immunocytochemistry Fluorescent immunohistochem istry Indirect immunocytochemistry Immunostain
Chapter  Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence micro scopy Fluorescent immunocytochemistry Fluorescent immunohistochem istry Indirect immunocytochemistry Immunostain

Chapter Antibodies Keywords Immunohistochemistry Antibody labeling Fluorescence micro scopy Fluorescent immunocytochemistry Fluorescent immunohistochem istry Indirect immunocytochemistry Immunostain - Description


Antibody Molecules Making Antibodies 10 Talking About Antibodies 13 Finding and Getting Antibodies 14 Choice of Primary 1 Antibodies 15 Antibodies Handling and Storing 16 Recommended Storage Freezer 20 16 Reco ID: 35911 Download Pdf

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Chapter2 Antibodies Keywords Immunohistochemistry · Antibodylabeling · Fluorescencemicro- scopy · Fluorescentimmunocytochemistry · Fluorescentimmunohistochem- istry · Indirectimmunocytochemistry · Immunostaining Contents Introduction ................................... 7 AntibodyMolecules ............................... 8 MakingAntibodies ................................ 10 TalkingAboutAntibodies ............................. 13 FindingandGettingAntibodies .......................... 14 ChoiceofPrimary(1  )Antibodies ......................... 15 AntibodiesHandlingandStoring ......................... 16 RecommendedStorageFreezer,Š20  C ..................... 16 RecommendedStorageRefrigerator,4  C .................... 16 Introduction Anantibody(Ab)isthekeyreagentofimmunocytochemistry.Touseantibodies effectively,considertheirstructure,function,andgeneration.Suchbasicknowledge aboutantibodiesisessentialtosucceedinidentifyingsuitableexperimentaldesign, Žndingantibodies,andtrouble-shootingproblems. Immunocytochemistrytakesadvantageofthreepropertiesofantibodies: 1.Antibodiesuniquelybindtoaproteinorothermolecule. 2.Antibodybindingtomoleculesisessentiallypermanentatphysiological conditions. 3.Newantibodiescanbemadetailoredtonewinterestingmolecules. 7 R.W.Burry, Immunocytochemistry ,DOI10.1007/978-1-4419-1304-3_2, C  SpringerScience+BusinessMedia,LLC2010 8 2Antibodies AntibodyMolecules Animmuneresponsegeneratesantibodiesorproteinscalled immunoglobulins(Igs) . AntibodiesarefurtherclassiŽedintomultiple isotypesorclasses (Table2.1).In immunocytochemistry,the IgG isotypeispreferredbecauseitsgenerationandbind- ingismoreconsistent.IgMantibodiescanbeusedifnootherisotypeisavailable. TheIgGmoleculescanbebrokendownintofour subclasses ,IgG1,IgG2,IgG3, andIgG4.Inimmunocytochemistryexperiments,thesesubclassesdonotmatterfor mostspeciesofanimals,buttheyareimportantforantibodiesgeneratedinmouse monoclonalantibodies(IgG1,IgG2a,IgG2b,andIgG3),aswewillseeinlater chapters. Table2.1 Igisotypes AntibodyisotypeAction IgAGut,respiratory,urinaryresponse IgDInitialimmunesystemresponse IgEResponsetoallergensŠhistaminerelease IgGImmuneresponsetoinvadingpathogens IgMEarlyimmuneresponseforpathogens Inusingantibodies,knowledgeoftheIgGstructureisimportant(Fig.2.1). IgG hasaconstantregionandavariableregion .The constantregion containsspecies- speciŽcsequencesandthe Fcportion thatbindsanFcreceptor(Fig.2.1,clearend), whichisfoundoncirculatingwhitecells,macrophages,andnaturalkillercells. TheFcportionalsohasspecies-speciŽcsitesthatareuniquetotheanimalspecies Fig.2.1 Theantibody.An IgGantibodyhasasingle constantregion( white )with theFcportionandthe species-speciŽcantigens.The variableregion( gray ) containstheFabportionthat bindstheepitopeportionof theantigen.Thesmall protein,onlyinthevariable region,isknownasthelight chain;thelargeproteinthatis partoftheconstantand variableregionistheheavy chain.TheIgGcanbe digestedbytheprotease enzyme,papain,intoanFc end(constantend)andaFab end(variableend)

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