Immunohistochemistry: Basics and Methods

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 colleaguesfromtheMünsterUniversityClinicforsharingprobesandreagents. IgorB. BuchwalowandWernerBöcker Münster 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.