英文免疫组化

英文免疫组化第1页，课件共21页，创作于2023年2月ProcedureIncubatetissuewithAbsoitbindstoantigenpresentinthetissueWashtissuesoonlyboundantibodyremainsExposetissuewithAbtosecondAbwhichrecognizes1stAb2ndAbisconjugatedtofluorescentchemicalorenzymesystemwhichformsavisiblereactionproduct第2页，课件共21页，创作于2023年2月MajorGoalMaximizesignal(positivestaining)Minimizebackgroundstaining(falsepositivestaining)第3页，课件共21页，创作于2023年2月ProcedureModificationAddNeutravidinwhichwillbeattractedtobiotinylatedsecondaryAb.NeutravidinwillbeconjugatedtothefluorescenttagAlexa488(greenfluorescenttag)orCY(redfluorescenttag)第4页，课件共21页，创作于2023年2月AbwithSpecificRecognitionCharacteristicsPrimaryAbraisedinrabbitSecondaryAbraisedagainstrabbitseruminsomeotheranimal(sheep,goat,orrodents)第5页，课件共21页，创作于2023年2月DoubleLabelingFordoublelabeling-primaryAbrasiedindifferentanimalsrabbitandratsincaseofdoublelabelingsecondaryAbhavedifferentmaterialsconjugated(anti-rabbitIgGconjugatedwithfluoresceinandanti-ratIgGconjugatedwithHRP)第6页，课件共21页，创作于2023年2月TissueAntigens2ndAb1stAb2ndAbconjugatedwitheitherfluorescentorenzymemarkerHRPorAlkalinePhosphatase第7页，课件共21页，创作于2023年2月DetailedProcedureTissuefixedTissuecutintothinnersections(15uM)Mountedongelatinorpoly-D-lysinesectionsUseofTritonXUseofserums(directedagainst2ndAb),drymilkproducts,avidin,H2O2第8页，课件共21页，创作于2023年2月ControlsBestprocedure-incubateprimaryAbwithantigen-antigenpreabsorptionOmittheprimaryAbinthe1stincubationstepUseapositivecontrol-usetissueknowntocontainantigenofinterest.第9页，课件共21页，创作于2023年2月HighBackgroundStaining1.Endogenousperoxidaseactivity-notcompletelyblocked.2.Non-specificbindingofproteintospecimen-needforblockers.3.Excessiveapplicationoftissueadhesive4.Inadequaterinsingofslides5.Improperantibodydilution6.Dryingoutoftissue.7.Overdevelopmentofsubstrate.第10页，课件共21页，创作于2023年2月PossibleCausesofNegativeStainingStepsforstainingnotperformedincorrectsequence.Eitherprimaryorsecondaryantibodyincubationstepswereskipped.DestructionoflabileantigensImproperfixationandorprocessingofsample第11页，课件共21页，创作于2023年2月PossibleCausesforWeakStainingSpecimenretainstoomuchliquidafterrinsingstepsIncubationtimesaretooshortorantibodysolutionistooweak.PoortiterofprimaryantibodyImpropersubstratepreparationoruseofoldsubstratesolution第12页，课件共21页，创作于2023年2月AntibodiesAb-proteinsoftheimmunoglobulinfamilythatbindtodifferentantigenmolecules.Immunoglobulinclasses(IgG,IgM,IgE)andsubclasses(IgG1,IgG2)AbareproducedbyB-lymphocytesoftheimmunesystem.EachcloneofB-lymphocytesisprogrammedtoproduceasinglespeciesofantibodywithsinglespecificityforantigen.第13页，课件共21页，创作于2023年2月PolyclonalAbscontAbsaremoleculespecificInjectanimalswithpureforeignmolecule(notusuallyseenbytheimmunesystemoftheanimal),animalproduceslargeamountsofantibodytothemolecule.Removebloodfromimmunizedanimalandseparateandpurifyformotherantibodies.第14页，课件共21页，创作于2023年2月PolyclonalAntibodiesPolyclonalreferstothefactthatmanydifferentantibodiesmayberaisedtodifferentpartsofthemolecule.Eachantibodyisproducedbyasinglecell.Manydifferentantibodiesareproducedtoasinglemoleculeandthesehavecomeeachfromdifferentcellclones第15页，课件共21页，创作于2023年2月ProcedureforPolyclonalsAbsInjectintorabbitsapurifiedmoleculesuchasaproteinorpeptidesshorterlengths)canbesynthesizedandinjected.Injectrabbitseveraltimesover2-3months.Separatetheantibodyofinterestusingsyntheticpeptidesplacedinacolumnandeludingthemoffthecolumn.

第16页，课件共21页，创作于2023年2月PolyclonalAntibodiesAbsaremoleculesproducedbyimmunesystemagainstinvadingmicroorganisms.EachantibodyisproducedbyasinglecellandthebodyinducesdivisionofthesecellstoincreaseAbproduction(clonesoforiginalcell).Onceantibodiesareboundtoinvadingorganism,specifiedcellsdestroytheantibody-boundmicrobe.第17页，课件共21页，创作于2023年2月MonoclonalAntibodiesMabsareproducedbyfusionofanimmortalizedmyeloma(cancer)celllinewithaB-lymphocytetoproduceahydridomacellline.Onceyouisolatethehybridomaclonewiththedesiredantigenspecificity,itispossibletoproduceanendlesssupplyofhighlyspecificantibody.第18页，课件共21页，创作于2023年2月AdvantagesofMabsKeyadvantage-specificityPolyclonalarecollectedfromserumofimmunizedanimalsandcontainamixtureofantibodyspeciesthatrecognizedifferentsubstructure(epitopes)withinthetargetantigenmoleculeAnotheradvantage-quantitySupplyofpolyclonalantibodiesarelimitedtoamountofserum第19页，课件共21页，创作于2023年2月SourcesofMabsRepositoriesincludeAmericanTypeCultureCollection/catalogs.htmlCommericalsuppliers$200-$500/20ugMSRSCatalogclaimstoprovideacomprehensiveinventoryofallcommerciallyavailablean