Immunohistochemistry

Visualization of proteins in tissues and cells by staining with antibodies

Methods

Immunohistochemical procedures are carried out on very thin tissue sections to detect a specific protein in brain tissue or in brain cells. In case of changes in gene expression, one expects a corresponding alteration in the amount of gene encoded protein. The latter can then be localized precisely with immunohistochemical tools. The principle of this procedure is based on the binding of an organic molecule specific to the surface structure (antigen) of the requested protein. When the antibody in addition is coupled to a dye, the distribution of the protein can be localized by microscopy.

The antibody-antigen interaction can be visualized in the following manner:

  • The avidin-biotin method (ABC), for example, uses a color-producing reaction to visualize a certain protein by light microscopy.
  • Immunofluorescence is another methodological option by which means the antibody is labeled with a fluorophore such as FITC, Texas Red, or rhodamine, etc.. Using fluoroscopic microscopy or confocal laser scanning microscopy, the distribution of the multiple proteins can be visualized at a cellular level.
Example for the ABC method: Even four week after cell implantation, the canula track is visible by due to induction of reactive astroglial cells. As the consequence of a traumatic stress response, astrocytes express the so-called glial fibrillary acidic protein (GFAP) which can be visualized by light-microscopy as a brown deposit.
Double fluorescence detection of astrocytes (red) and capillaries (green) in cortex of the rat using antibodies with different fluorophores. Astrocytes are cells with multiple processes which are in contact with endothelial cell (as well as neurons, not shown). This guarantees an optimal supply of nutrients, i.e., glucose and oxygen.

Technical equipment

Apart from light microscopy, fluorescence microscopy is an important tool: instead of visible light, the UV-range is used for the illumination of brain sections. This allows the visualization of fluorescent dyes using adequate optical filters. The specimen is illuminated with light at a specific wavelength which is absorbed by the fluorophores, causing them to emit longer wavelengths of light. The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Most fluorescence microscopes are epi-fluorescence microscopes, i.e., excitation and observation of the fluorescence occur from above the specimen).

A further development of light and fluorescence microscopy is the so-called confocal Laser-Scanning-Microscopy (LSM). Instead of optics a fine laser beam raster scan is used to sample the fluorescent light information within a brain section allowing not only a 2-D but also a 3-D assessment of fluorophore distribution and thus of protein identification within the same cell. When the various antibodies are labeled with different fluorophores the various cell types can be spatially attributed (see above).

 

Detection of embryonic stem cells within the infarct border zone following experimental cerebral stroke in rat.

Confocal Laser-Scanning-Microscope (LSM) from LEICA (SP2) with different laser sources for the illumination of fluorophores.


See also Microscopy