Chapter 45 : Immunohistochemistry: Principles and Advances

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This chapter presents current, practical methods that are applicable for performance of quality immunostains in the modern automated immunohistochemistry (IHC) laboratory. The cross-links are believed to be responsible for masking antibody-binding epitopes and to be a major cause of lack of sensitivity in paraffin section IHC. In spite of its inherent detrimental effects on antigenicity and IHC, a proper and consistent protocol for fixation with neutrally buffered formalin is imperative for high-quality IHC. Paraffin tissue sections for IHC are usually cut at a thickness of 4 to 6 μm. In order to retain sections on the glass microscope slide throughout the staining process, it is necessary to use slides that have been specially treated to increase their adhesive properties. Most antibodies used in a clinical IHC laboratory are commercially available and have well-characterized specificities. For analyte-specific reagents, it is assumed that good manufacturing practices have been followed and that the antibody will likely perform as advertised. Antigen retrieval enables antibodies that might previously have performed only in frozen tissues to be reactive in fixed tissues and has greatly expanded the number of antibodies that can now be applied to paraffin-embedded tissue.

Citation: Roche P, Hsi E, Firfer B. 2006. Immunohistochemistry: Principles and Advances, p 396-402. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch45
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Protocol for preparation of 10% paraformaldehyde stock solution

Citation: Roche P, Hsi E, Firfer B. 2006. Immunohistochemistry: Principles and Advances, p 396-402. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch45
Generic image for table

Protocol for steam heat-induced antigen retrieval

Citation: Roche P, Hsi E, Firfer B. 2006. Immunohistochemistry: Principles and Advances, p 396-402. In Detrick B, Hamilton R, Folds J (ed), Manual of Molecular and Clinical Laboratory Immunology, 7th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815905.ch45

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