Combiningtwodifferentscienti?cdisciplines-morphologyandimmunochemistry-
immunohistochemistryhasdevelopedasanimportantinstrumentinresearchand
clinicalpathology. A basicunderstandingofunderlying
principlesandpotential
problemsisunavoidableifyouwanttobesuccessfulinyouruseofimmunohis-
chemistry,aswellasingettingyourpaperspublishedandyourresearchgrants
funded.
Whilemanyexcellenttextsandmonographsexistwhichcovervariousaspects
ofimmunohistochemistry,thelackofaconcisecomprehensiveguidetousing
thesemethodswasamajormotivationforwritingthisbook.
Ourintentionwasto
createaneasy-to-readandfocusedresourcebasedonstate-of-the-artinformation
forabroadaudiencerangingfromstudentsandtechnicalassistantstoexperienced
researchers.
Thishandbookhasaconciseformat,withprotocolsandinstructionsfor
methodsimmediatelyfollowingtheshortintroductorytheoreticalmaterialineach
chapter.
BeingconsciousofthegrowingroleofInternetasaninformationsource,
wehavefounditreasonableinmanycasestosubstitutecitingbooksandjournal
publications with corresponding Internet websites. Where possible,
commercial
sourcesofreagents,kits,andequipmentarelistedthroughoutthetextinsteadof
inaseparateindex.
Thougheachchapterissmallandintroductory,thishandbook itself is
self-suf?cient and provides a comprehensive look at the principles
of immunohistochemistry. For readers wanting further depth of
knowledge, each
chapterisbackedupbyashortlistofcarefullyselectedoriginalarticles.
Duringthelastdecade,pioneeringeffortsofhistochemistshaveledtoan-
menseimprovementinthereagentsandprotocols.
Theresearcherisurgedalwaysto
determinethereasonforeverymethodandstepbeforedoingit.
Thishandbookis
intendedtohelpreaderstoavoidtroublesinthechoiceofanadequatemethod,which
happenswhenusingstandardtextbooks.
Forthishandbook,wecarefullyselected
establishedmethodsandeasy-to-adoptprotocols,payingattentiontomoderndev-
opmentsinimmunohistochemistry,suchasantigenretrieval,signalampli?cation,
the use of epitope tags in immunohistochemistry, multiple
immunolabeling or diagnosticimmunohistochemistry.
Eachofthemethodsdescribedinthishandbook v vi Preface was provedby
the authors; many of these methods are routinely used in daily
practiceintheirinstitute.
Allthepracticalmethodsadvocatedareclearlydescribed,
withaccompanyingtables,andtheresultsobtainableareillustratedwithcolour
micrographs. Acknowledgements We thank Vera Samoilova forthe
perfect technical assistance and other
colleaguesfromtheMunsterUniversityClinicforsharingprobesandreagents.
IgorB. BuchwalowandWernerBocker Munster Contents 1
AntibodiesforImmunohistochemistry ...1 1. 1 StructureofAntibodies
...2 1. 2 PolyclonalAntibodies ...4 1. 3 MouseMonoclonalAntibodies
...4 1. 4 RabbitMonoclonalAntibodies ...5 1. 5
ProteinAandProteinGinImmunohistochemistry ...7 References ...8 2
AntibodyLabelingandtheChoiceoftheLabel ...9 2. 1
CovalentLabelingofAntibodies ...9 2. 2
Non-CovalentLabelingofPrimaryAntibodieswith LabeledFabFragments
...10 2. 3 EnzymeLabelsforLightMicroscopy ...13 2. 4
FluorophoreLabelsforFluorescenceMicroscopy ...15 2. 5
ColloidalGoldLabelsforElectronMicroscopy ...16 References ...17 3
ProbesProcessinginImmunohistochemistry ...19 3. 1
FixationinImmunohistochemistry ...19 3. 1. 1
FixationinAlcoholsandAcetone ...20 3. 1. 2 FixationinFormaldehyde
...20 3. 1. 3 EffectofFormaldehydeFixationon Antigen-nster Contents
1 AntibodiesforImmunohistochemistry ...1 1. 1 StructureofAntibodies
...2 1. 2 PolyclonalAntibodies ...4 1. 3 MouseMonoclonalAntibodies
...4 1. 4 RabbitMonoclonalAntibodies ...5 1. 5
ProteinAandProteinGinImmunohistochemistry ...7 References ...8 2
AntibodyLabelingandtheChoiceoftheLabel ...9 2. 1
CovalentLabelingofAntibodies ...9 2. 2
Non-CovalentLabelingofPrimaryAntibodieswith LabeledFabFragments
...10 2. 3 EnzymeLabelsforLightMicroscopy ...13 2. 4
FluorophoreLabelsforFluorescenceMicroscopy ...15 2. 5
ColloidalGoldLabelsforElectronMicroscopy ...16 References ...17 3
ProbesProcessinginImmunohistochemistry ...19 3. 1
FixationinImmunohistochemistry ...19 3. 1. 1
FixationinAlcoholsandAcetone ...20 3. 1. 2 FixationinFormaldehyde
...20 3. 1. 3 EffectofFormaldehydeFixationon Antigen-
immunohistochemistryhasdevelopedasanimportantinstrumentinresearchand
clinicalpathology. A basicunderstandingofunderlying
principlesandpotential
problemsisunavoidableifyouwanttobesuccessfulinyouruseofimmunohis-
chemistry,aswellasingettingyourpaperspublishedandyourresearchgrants
funded.
Whilemanyexcellenttextsandmonographsexistwhichcovervariousaspects
ofimmunohistochemistry,thelackofaconcisecomprehensiveguidetousing
thesemethodswasamajormotivationforwritingthisbook.
Ourintentionwasto
createaneasy-to-readandfocusedresourcebasedonstate-of-the-artinformation
forabroadaudiencerangingfromstudentsandtechnicalassistantstoexperienced
researchers.
Thishandbookhasaconciseformat,withprotocolsandinstructionsfor
methodsimmediatelyfollowingtheshortintroductorytheoreticalmaterialineach
chapter.
BeingconsciousofthegrowingroleofInternetasaninformationsource,
wehavefounditreasonableinmanycasestosubstitutecitingbooksandjournal
publications with corresponding Internet websites. Where possible,
commercial
sourcesofreagents,kits,andequipmentarelistedthroughoutthetextinsteadof
inaseparateindex.
Thougheachchapterissmallandintroductory,thishandbook itself is
self-suf?cient and provides a comprehensive look at the principles
of immunohistochemistry. For readers wanting further depth of
knowledge, each
chapterisbackedupbyashortlistofcarefullyselectedoriginalarticles.
Duringthelastdecade,pioneeringeffortsofhistochemistshaveledtoan-
menseimprovementinthereagentsandprotocols.
Theresearcherisurgedalwaysto
determinethereasonforeverymethodandstepbeforedoingit.
Thishandbookis
intendedtohelpreaderstoavoidtroublesinthechoiceofanadequatemethod,which
happenswhenusingstandardtextbooks.
Forthishandbook,wecarefullyselected
establishedmethodsandeasy-to-adoptprotocols,payingattentiontomoderndev-
opmentsinimmunohistochemistry,suchasantigenretrieval,signalampli?cation,
the use of epitope tags in immunohistochemistry, multiple
immunolabeling or diagnosticimmunohistochemistry.
Eachofthemethodsdescribedinthishandbook v vi Preface was provedby
the authors; many of these methods are routinely used in daily
practiceintheirinstitute.
Allthepracticalmethodsadvocatedareclearlydescribed,
withaccompanyingtables,andtheresultsobtainableareillustratedwithcolour
micrographs. Acknowledgements We thank Vera Samoilova forthe
perfect technical assistance and other
colleaguesfromtheMunsterUniversityClinicforsharingprobesandreagents.
IgorB. BuchwalowandWernerBocker Munster Contents 1
AntibodiesforImmunohistochemistry ...1 1. 1 StructureofAntibodies
...2 1. 2 PolyclonalAntibodies ...4 1. 3 MouseMonoclonalAntibodies
...4 1. 4 RabbitMonoclonalAntibodies ...5 1. 5
ProteinAandProteinGinImmunohistochemistry ...7 References ...8 2
AntibodyLabelingandtheChoiceoftheLabel ...9 2. 1
CovalentLabelingofAntibodies ...9 2. 2
Non-CovalentLabelingofPrimaryAntibodieswith LabeledFabFragments
...10 2. 3 EnzymeLabelsforLightMicroscopy ...13 2. 4
FluorophoreLabelsforFluorescenceMicroscopy ...15 2. 5
ColloidalGoldLabelsforElectronMicroscopy ...16 References ...17 3
ProbesProcessinginImmunohistochemistry ...19 3. 1
FixationinImmunohistochemistry ...19 3. 1. 1
FixationinAlcoholsandAcetone ...20 3. 1. 2 FixationinFormaldehyde
...20 3. 1. 3 EffectofFormaldehydeFixationon
Antigen-AntibodyBinding ...21 3. 2
Paraf?nSectionsforImmunohistochemicalAnalysis ...22 3. 2. 1
EmbeddingandCutting ...22 3. 2. 2 MountingParaf?nSectionsontoSlides
...23 3. 3 CryosectionsforImmunohistochemicalAnalysis ...24 3. 4
BuffersforWashingandAntibodyDilution ...25 3. 5
MountingFollowingImmunohistochemicalStaining ...27 3. 6
StorageFollowingImmunohistochemicalStaining ...28 References ...28
vii viii Contents 4 WorkingwithAntibodies ...31 4. 1
DirectImmunostainingMethod ...31 4. 2 IndirectImmunostainingMethod
...33 4. 3 TheChoiceofAntibodies ...35 4. 3. 1
TheChoiceofPrimaryAntibodies ...35 4. 3. 2
TheChoiceofSecondaryAntibodies ...
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