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Advanced Histological Techniques

Antibody Binding

The antibody systems used in Immunohistochemistry can be broadly divided into two types, direct and indirect. With the direct method the visualizing agent is attached directly to the antibody that will bind with the antigen. The direct method is technically and theoretically straightforward, and yields results sufficient for many studies. Its sensitivity is limited by the fact that only one antibody, and therefore one visualization agent, is bound to each antigen.

Labeled Antibody
With direct immunohistochemistry, the labeled antibody directly binds to the antigen.

Indirect immunohistochemistry utilizes a second antibody specific to the first (primary) antibody. This secondary antibody has the visualizing agent attached. This allows for signal amplification. Primary antibodies directly label the antigen. The secondary antibody binds to the constant region of each primary antibody, with many secondary antibodies binding to each primary. This creates a cascade effect, amplifying the signal.

Amplified Signal
The signal can be greatly amplified with indirect immunohistochemistry, in which the visualizing agent (horseradish peroxidase) is attached to polyclonal secondary antibodies with polyclonal primary antibodies binding antigen.


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