Immunohistochemistry and Immunocytochemistry: Essential Methods

241 Pages·2017·10.198 MB·English

by  Simon Renshaw

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Immunohistochemistry and
Immunocytochemistry
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Immunohistochemistry
and Immunocytochemistry
Essential Methods
EDITED BY
(cid:2) SIMON RENSHAW (cid:2)
Second Edition
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Thiseditionfirstpublished2017©2017byJohnWiley&Sons,Ltd.
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1 2017
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Table of Contents
ListofContributors vii
Preface ix
Acknowledgements xi
Chapter1:AntibodiesforImmunochemistry 1
MarkCooperandSheridenLummas
Introduction 1
ImmunogensforAntibodyProduction 5
AntibodyProduction 12
AntibodyPurification 16
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AntibodyLabelling 21
AntibodyStabilityandStorage 23
References 24
Chapter2:TheSelectionofReporterLabels 25
JudithLangenick
Introduction 25
EnzymaticLabels 26
FluorescenceDetection 29
References 32
Chapter3:ImmunohistochemistryandImmunocytochemistry 35
SimonRenshaw
SpecimenFormatsforImmunochemistry 36
Fixation 37
ProcessingTissueBlockstoParaffinWax 46
Microtomy 47
TissueMicroarrays 47
SpecimenStorage 48
Decalcification 49
AntigenRetrieval 50
Controls 56
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vi TableofContents
ImmunochemicalStainingTechniques(OptimizingaNewAntibody) 57
Counterstains 71
Mounting 74
Troubleshooting 76
ExamplesofImmunostainingPhotomicrographs 76
Acknowledgements 101
References 101
Chapter4:MultipleImmunochemicalStainingTechniques 103
SofiaKoch
Introduction 103
MethodsandApproaches 115
References 122
Chapter5:QualityAssuranceinImmunochemistry 123
PeterJacksonandMichaelGandy
Introduction 123
MethodsandApproaches 125
AutomatedImmunochemicalStaining 147
Troubleshooting 149
References 154
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Chapter6:AutomatedImmunochemistry 157
EmanuelSchenckandSimonRenshaw
Introduction 157
MethodsandApproaches 160
OtherFormsofAutomation 164
References 168
Chapter7:ConfocalMicroscopy 169
AnnWheeler
Introduction 169
WhenShouldConfocalbeUsed? 173
Applications:ForExampleCo-localization,Quantification,3DVisualization
andKinetics 173
HowToSetUpaConfocalExperiment? 174
References 198
FurtherReadings 198
Chapter8:UltrastructuralImmunochemistry 199
JeremySkepperandJanetPowell
Introduction 199
MethodsandApproaches 207
References 222
Index 227
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List of Contributors
MarkCooper
Abcamplc,Cambridge,UK
MichaelGandy
TheDoctorsLaboratoryLtd,London,UK
PeterJackson
DepartmentofHistopathology,LeedsGeneralInfirmary,Leeds,UK(retired)
SofiaKoch
Abcamplc,Cambridge,UK
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JudithLangenick
AbDSerotec,Oxford,UK
SheridenLummas
Abcamplc,Cambridge,UK
JanetPowell
CambridgeAdvancedImagingCentre,DepartmentofAnatomy,
UniversityofCambridge,Cambridge,UK
SimonRenshaw
Abcamplc,Cambridge,UK
EmanuelSchenck
MedimmuneLLC,Gaithersburg,MD,USA
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viii ListofContributors
JeremySkepper
CambridgeAdvancedImagingCentre,DepartmentofAnatomy,UniversityofCambridge,
Cambridge,UK
AnnWheeler
InstituteofGeneticsandMolecularMedicineAdvancedImagingResource,Universityof
Edinburgh,Edinburgh,UK
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Preface
I
MMUNOCHEMISTRY IS AN INVALUABLE TOOL forthevisualizationofcellular
antigensindiagnosticandbiologicalresearchenvironments.Theneedtoobtainaccu-
rate,reliableandreproducibleresultsisofparamountimportance.
ItiswiththisfundamentalaiminmindthatwehavecompiledImmunohistochemistry
and Immunocytochemistry: Essential Methods. We have achieved this by examining each
aspect of immunochemistry in turn, with each chapter including detailed information
regardingthesubjectmatterinquestion.Eachchapteriswrittenbyanexpertintheirfield
andincludesprotocolsthataretypicallyusedintheirownresearch.Inaddition,benefits
andlimitationsofeachapproacharediscussedwithinthechapters.
Thisbookoffersawealthofknowledgetothenoviceimmunochemist,who,fromthe
outset, wishes to fully understand the theory and practice of immunochemical staining
(cid:2) techniquesandobtainreliableandreproducibledatatimeandtimeagain.Fortheexpe- (cid:2)
riencedimmunochemist,thisbookisacomprehensivereferenceguidetothetheoryand
practiceofimmunochemicalstainingtechniques,allowingfurtheroptimizationofexisting
immunochemicalstainingprotocols.
SimonRenshaw
January2017
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Acknowledgements
T
HANK YOU TO ALLofmyfriends,familyandcolleaguesforyourcontinuedsup-
portthroughoutthisproject.
Aspecialthankyougoestothecontributingauthors,withoutwhomthisbookwould
havetakenconsiderablylongertowrite!
ThankyoutoElsevierLtd,AbcamplcandLeicaBiosystemsforkindlyagreeingtorepro-
ductionofcopyrightedmaterials.
Finally,averyspecialdedicationgoestoChrisvanderLoos,whohadverykindlyagreed
tobetheauthorofthe‘MultipleImmunochemicalStainingTechniques’chapter,butsadly
passedawaybeforebeginningthework.Hewasincrediblygiftedinhisfieldanddelivered
amostinformativeandentertaininglecture.Hewillbemissedbymany.
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CHAPTER ONE
Antibodies for Immunochemistry
Mark Cooper and Sheriden Lummas
Abcamplc,Cambridge,UK
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INTRODUCTION
Unlikeinnateimmunity,theadaptiveimmuneresponserecognizes,reactstoandremem-
bersforeignsubstancesinvadinganorganism.Antibodiesplayacentralroleinthefunction
ofadaptiveimmunity.Theirrolesaretodetect,specificallybindandfacilitatetheremoval
offoreignsubstancesfromthebody.MemoryBcellscreateanimmunologicalmemorythat
allowstheimmunesystemtorespondquickeruponsubsequentexposuretothesamefor-
eignsubstance.
A substance not recognized by the immune system as being native to the host and
therefore stimulates an immune response is known as an antigen (antibody generator).
Bindingofanantigentoanantibodyisspecific.Biochemicalresearchutilizestheabilityof
antibodiestodistinguishbetweenantigensandtodetectbiologicalmolecules(commonly
proteins)incellsandtissuesusingimmunochemicalstainingtechniques.Immunochem-
istryisthefocusofthistext,anditspracticeisdiscussedindetailthroughoutlaterchapters
(seep35).
ImmunohistochemistryandImmunocytochemistry:EssentialMethods,SecondEdition.EditedbySimonRenshaw.
©2017JohnWiley&Sons,Ltd.Published2017byJohnWiley&Sons,Ltd.
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