Immunohistochemistry

Immunohistochemistry or IHC is a method for labeling specific cells by utilizing antibodies to bind specific antigens (proteins) in biological tissues.

IHC provides a way to assess the localization and distribution of different proteins in different tissues.  IHC also preserves the extracellular environment of the tissues, so that spatial relationships and structural components can be observed as well.

Utilizing molecular markers that are characteristic of certain cell types or events, it is possible to stain individual cells.


1) Ganglion in the myenteric plexus                                    2) Mature Neurons

In order to visualize the interaction between antibody and antigen, the antibody is either conjugated (chemically linked) to an enzyme that catalyzes a color-producing reaction OR the antibody itself is tagged to a flourophore (functional group that fluoresces in response to certain light absorption).

There are also two methods of IHC: DIRECT and INDIRECT.


3) Glial cell supporting neurons

Indirect IHC, the most common form of IHC,  begins with an unlabeled primary antibody that binds the antigen, however, a secondary antibody which is labeled, is added in order to bind to the primary antibody.  Since multiple secondary antibodies bind the primary antibody, this greatly amplifies the signal.

PROCEDURE

First, the tissue must be preserved and sliced into sections that are each several micrometers thick.  The paraffin used to preserve the tissue must also be washed from the slides, and the rehydrated with a buffer such as PBS.

Next, antigen retrieval is necessary if formalin was used to fix the tissue.  This step breaks methylene bridges that have formed, thus exposing the antigenic sites of the tissue where the antibodies will bind.  Endogenous enzymes must be blocked so as not to interfere with the binding, and another wash is performed.

A protein-blocking step precedes the addition of the primary antibody.  Another wash removes any nonspecific binding, and then the secondary antibody is added (link antibody).  This antibody can be labeled before, or after it is added depending on the protocol.  Chromogen can be added to provide the dye, and a counterstain can be added to provide a contrast when visualizing the final stained slide.

This video below provides a short synopsis of the method:

[kml_flashembed movie="http://www.youtube.com/v/OQz6iMDBYEw" width="640" height="385" allowfullscreen="true" fvars="fs=1" /]

This procedure is the best means for viewing neurogenesis.  By utilizing specific markers, or in conjuction with a label of proliferating cells, such as BrdU, it possible to assess different cell types at points in the cell’s life cycle.  The prepared slides are viewed using a confocal microscope.

References:

http://www.flickr.com/search/?q=immunohistochemistry

http://www.ihcworld.com/introduction.htm

http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-application/protocols/immunohistochemistry.html

http://en.wikipedia.org/wiki/Immunohistochemistry

By Avi Goch

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