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Cytology · Year 1 · Medical University of Sofia

06

Immunohistochemistry

Free notes for topic 06 of the Cytology syllabus, open without an account. Written by a senior student against the syllabus question and checked line by line by a second student before publishing. How content is made

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Summary

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Immunohistochemistry is the process of using antibodies to identify the presence of antigens in a section. Where the other histochemical methods detect a whole class of molecule, this one detects a single named protein, because that is what an antibody binds.

Two things have to be chosen. The antibody: polyclonal, from different clones of plasma B cells and binding different epitopes, or monoclonal, from a single clone and binding one particular epitope. And the method: direct, where the label sits on the primary antibody, or indirect, where a labelled secondary antibody binds the primary one.

Indirect is the one used in practice, and the reason is worth remembering: more than one labelled secondary antibody can bind each primary antibody, so the signal is amplified.

1. What immunohistochemistry is

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Immunohistochemistry is the process of using antibodies to identify the presence of antigens.

Antibodies belong to the immunoglobulin family of glycoproteins.

The closely related technique immunocytochemistry is used to determine the localisation of specific proteins or antigens.

2. Types of antibodies

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There are two types of antibody, and they differ in how many clones of cell produced them.

Polyclonal antibodies are produced by different clones of plasma B cells. They interact with different epitopes on the same antigen.

Monoclonal antibodies are produced by the same clone of plasma B cells. They interact with a particular epitope on the same antigen. Their advantage is highly specific binding.

3. Labelling the antibody

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The process runs as follows:

  • The tissue section, carrying the antigen, is placed in a solution containing the antibody.
  • The antibody binds to the antigen.
  • After a rinse, the antigen's location can be seen under a light or electron microscope by visualising the antibody.

An antibody on its own is invisible, so it has to carry a label. Antibodies are commonly coupled with:

  • a fluorescent compound, giving the immunofluorescence method;
  • an enzyme, peroxidase, giving the PAP method;
  • gold particles, for the transmission electron microscope.

4. Two methods of localising antigen

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The two methods differ in whether the label is carried by the antibody that binds the antigen, or by a second antibody that binds the first.

Diagram comparing direct immunofluorescence, where a labelled antibody binds the antigen directly, with indirect immunofluorescence, where an unlabelled primary antibody binds the antigen and several labelled secondary antibodies bind the primary
Diagram comparing direct immunofluorescence, where a labelled antibody binds the antigen directly, with indirect immunofluorescence, where an unlabelled primary antibody binds the antigen and several labelled secondary antibodies bind the primary

Direct immunohistochemistry

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Direct immunohistochemistry uses a primary antibody with a label, for example a fluorescent compound. It binds specifically to the antigen.

  • Advantageit needs only the primary antibody, so it is faster and less complex.
  • Disadvantagelow intensity.

Indirect immunohistochemistry

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Indirect immunohistochemistry was devised to improve intensity.

The primary antibody binds to the antigen. Then a labelled secondary antibody, carrying for example a fluorescent compound or peroxidase, binds to the primary antibody, indirectly labelling the antigen of interest.

Because more than one labelled secondary antibody can bind each primary antibody, the sensitivity and signal intensity are much greater.

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