Biomedical Engineering Reference
In-Depth Information
Table 2
Main steps on standard IHC protocol
Step
Material
Purpose
Antigen retrieval
Enzymatic (proteases) or heat-based
(microwave or laboratory steamer)
Re-expose antigens after formalin
fixation
Endogen peroxidase
blocking
Incubation with hydrogen peroxide
Avoid nonspecific staining due to
endogenous peroxidase
Primary antibody
incubation
Antibody directed against specific antigen Recognize antigens and initiate the
IHC reaction
Secondary antibody
(detection system)
Avidin-biotin or peroxidase-
antiperoxidase-based reactions
Label the immune reaction with an
enzymatic reporter molecules
Chromogens
Peroxidase and alkaline phosphatase
Reveal the reaction for light
microscopy
Counterstain
Toluidine blue or H&E
Lightly stain background tissue
are the low numbers of antigens that are optimized for this retrieval
method, the possibility of morphological alterations on the tissues
and epitope destruction [
95
]. The heat-induced epitope retrieval
methods are the most commonly used and rely on high tempera-
tures, created by a microwave oven or a laboratory steamer, to
partially reverse the chemical reactions between protein and for-
malin [
95
,
101
].
Along with tissue fixation, the choice of the primary antibodies is
the most important factor for success of an IHC protocol. Anti-
bodies are made by immunizing animals (usually mouse, rabbit,
goat, or horse) with purified antigens. Those who are produced in
multiple species are called polyclonal and those produced in only
one species (usually mouse) are called monoclonal [
96
]. Polyclonal
antibodies present the advantage of identifying multiple epitopes of
the desired antigen, thus increasing the chance of reaction, but
present the disadvantage of an increased likelihood of nonspecific
cross-reactivity with similar antigens, causing false-positive reac-
tions [
95
,
96
]. Polyclonal antibodies (which in fact are an antise-
rum) also contain multiple macromolecules that cause more intense
nonspecific background staining when compared to monoclonal
antibodies. Monoclonal antibodies are highly specific for a single
epitope of an antigen thus the possibility of cross-reactivity is
markedly reduced. On the other hand monoclonal antibodies
might pose problems in reacting to “hard to detect” antigen in
fixed tissues [
95
].
The availability of effective antibodies for specific antigens
should be thoroughly researched either on the literature or with
commercial sources. Manufacturer's data is usually a good point for
3.2.2
Primary Antibodies
