英文免疫組化

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

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