Making

Immunohistochemistry techniques have been used since the 1940s, when AH Coons and colleagues published their landmark paper describing the identification of tissue antigens using a direct fluorescence protocol (Coons AH, Creech HJ, Jones RN (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202).

Immunohistochemistry enables the visualization (using light or confocal microscopy) of the tissue distribution of specific antigens (or epitopes). The process localizes protein targets of interest by applying specific monoclonal or polyclonal antibodies to tissue surfaces in a process called antibody incubation. In recent years, the protocol has been refined with the advent of avidin-biotin immunohistochemistry.

This procedure makes use of the high affinity that avidin (or streptavidin) has for biotin. Multiple binding sites on the molecules result in the amplification of the tissue signal which is useful when trying to detect low copy number antigens.

A collection of useful immunohistochemistry protocols and other methods are available in the protocols section of the website. Protocol optimization is crucial to obtaining successful and reproducible results; the troubleshooting section has some useful suggestions for improving immunohistochemistry results.

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histochem.net is an immunohistochemistry information resource for researchers. Please follow the all safety and precaution recommendations supplied with the products or equipment you use as well as your institutional guidelines. The protocols and suggestions on this website are not a substitute for formal training.

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Unique experiences to stain slides


 

avidin - biotin & dab immunohistochemistry
cryosectioning 
sample fixation 
antigen retrieval 
mounting 
periodic acid Schiff 

 

 

 

Exercise caution. Please follow the manufacturer’s recommendations and instructions when working with any reagent or system. The procedures posted on this website are for informational purposes only.

 


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Immhunohistochemistry Terms and Definitions 

antigen


antigens are typically proteins, however, they can be any substance that triggers an immune response and antibody production


antigen retrieval is a protocol to expose antigens masked by fixation (see antigen retrieval protocol)

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avidin - biotin complex


there is a high affinity between avidin and biotin; immunohistochemistry makes use of this high affinity by using biotinylated antibodies to amplify tissue antigen signals

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counterstain


after immunohistochemical processing, counterstains are often applied to better visualize the results

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cryosection


cryosectioning is a tissue sectioning technique that cuts frozen tissue using a cryostat (a microtome operating at approximately -25 C)

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dewax


the process of removing paraffin from paraffin embedded tissues

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fixative


a solution that helps preserve tissue and cell morphology

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IgG


immunoglobulin G - an antibody type normally present in the blood

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IgM


immunoglobulin M - one of the 5 classes of immunoglobulins

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primary antibody


primary antibodies are glycoproteins that specifically bind antigens (epitopes)

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secondary antibody


a secondary antibody is raised to the Ig of the species from which comes the primary antibody; the secondary antibody will label the primary antibody

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triton


triton (triton x-100) is a nonionic surfactant; it is used to permeabilize cell memnranes to allow greater access to antibodies

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