Biomedical Engineering Reference
In-Depth Information
starting. Many aspects need to be accounted while choosing an
antibody. Among them are the species in which it was developed
and species it is intended for (e.g., Rabbit-anti human), possible
species cross-reactivity (antibodies specific for humans usually cross
react and can be used in other species like non-human primates or
dogs), application intended (IHC, Western blotting, Flow cytome-
try), methods of tissue preparation tested (frozen or fixed tissue),
clone of the antibody, etc. Once a commercial relationship is
established manufacturers can be helpful
in troubleshooting
technical issues.
In order for the antigen-antibody reaction to be visualized by
light microscopy it needs to be labeled with a visible molecule.
Detection systems allow the labeling of the immune reaction by
attaching fluorescent or color-expressing enzymatic reporter
molecules to the primary and secondary antibodies [
95
,
96
].
Detection methods can be classified as direct or indirect. Direct
methods are composed of a one-step process with the antibody
conjugated with a reporter molecule [
95
,
102
]. Indirect methods
are more sensitive and are characterized by the presence of a
secondary labeled antibody that presents high affinity with the
primary antibody. This renders the primary antibody unlabeled,
retaining its original conformation and activity and results in a
stronger signal with larger number of antigen-antibody bindings
[
96
,
102
]. Common indirect methods are the avidin-biotin meth-
ods (avidin-biotin complex [ABC], labeled streptavidin-biotin
[LSAB]), and the peroxidase-antiperoxidase (PAP) method)
[
102
]. Another indirect method called polymeric labeling two-
step method (EnVision
3.2.3 Detection Systems
) presents a simpler,
although more expensive, alternative to ABC or LSAB methods
with similar if not higher sensitivity and lack of background stain-
ing. It consists of a polymer that harbors multiple molecules of
enzymes and the secondary antibody that binds directly to the
primary antibody [
103
,
104
].
After the secondary antibody is incubated the reaction needs
to be revealed by an enzyme-chromogen reaction. The most
commonly used enzymes are peroxidase and alkaline phosphatase
and the most commonly used chromogens are 3,3
0
diaminoben-
zidine tetrachloride (DAB) that imparts a brown color to the
reaction, 3-Amino-9-ethylcarbazole (AEC) that gives a red
color, and 4-Chloro-1-naphthol that causes a blue reaction
[
102
]. DAB is the most popular chromogen but if the IHC
protocol targets highly pigmented (particularly with melanin)
tissues like the uvea this chromogen should be avoided (due to
its brown color) and replaced by a chromogen that results in a
more contrasting color, like AEC (red) or 4-Chloro-1-naphthol
(blue) [
78
,
95
,
96
,
105
].
,PowerVision
™
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