Immunohistochemistry: Basics and Methods

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 colleaguesfromtheMu¨nsterUniversityClinicforsharingprobesandreagents. IgorB. BuchwalowandWernerBo¨cker Mu¨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–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 . . . . . . . . . . . . . . . . . . . . . . . . .