Intro to Ihc

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Techniques of

Immunohistochemistry:

Principles, Pitfalls, and

Standardization

Clive R. Taylor  •  Shan-Rong Shi  •  Nancy J. Barr

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Introduction  1

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Techniques, Protocols, and “Troubleshooting”  22

Basic Principles of Immunohistochemistry  2

Antibodies as Specific Staining Reagents  2

Blocking Non-specific Background Staining  4

Detection Systems  5

Quality Control and Standardization  13

Tissue Fixation, Processing, and

Antigen-Retrieval Techniques  18

INTRODUCTION

Immunohistochemistry (IHC), or ­mmunocytochemistry,

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is a method for localizing specific antigens in tissues or

cells based on antigen-antibody recognition; it seeks to

exploit the specificity provided by the binding of an antibody with its antigen at a light microscopic level. IHC has

a long history, extending more than half a century from

1940, when Coons developed an ­mmunofluorescence

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technique to detect ­ orresponding antigens in frozen

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­ issue sections.1 However, only since the early 1990s

has the method found general application in surgical

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­ athology.2-5 A series of technical developments led

eventually to the wide range of IHC applications in use

today. The enzymatic label (horseradish ­ eroxidase),

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developed by Avrameas6 and by Nakane and ­ olleagues,7

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allowed visualization of the labeled antibody by light

microscopy in the presence of a suitable colorogenic

substrate ­ ystem. In Oxford, ­ aylor and Burns devels

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oped the first successful ­ emonstration of antigens in

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routinely processed ­ ormalin-fixed paraffin-embedded

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(FFPE) tissues.5 A critical issue in the early ­ evelopment

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of ­mmunoperoxidase techniques was related to the

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need to achieve greater sensitivity. Greater sensitivity would facilitate staining of FFPE ­ issues—from a

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­ imple ­ ne-step direct conjugate method to ­ ultiple-step

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m

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­ etection techniques such as the ­ eroxidase antiperoxip

dase (PAP),...