Histochemistry is the use of dyes and stains to demonstrate structures in thin sections of tissue.

In traditional histochemistry, tissues removed from the body are fixed in formalin (a buffered solution of formaldehyde), dehydrated in a progressively stronger series of alcohols and the solvent xylene and then set in paraffin wax. The wax supports the tissue as it is cut into thin sections (~4µm thick) by a microtome. Once the sections are placed on microscope slides, the wax is removed using xylene and the sections are rehydrated using a series of progressively less strong ethanol solutions. The sections can then be stained to demonstrate tissue structure and components.

Some tissue components are removed or damaged during the fixation and dehydration / rehydration process. In these cases, fresh, unfixed tissue may be snap frozen in a mounting medium and sections using a cryomicrotome which keeps the tissue and sections at -20°C or below. The disadvantage of this method is that tissues must be frozen rapidly to avoid autolysis (spontaneous breakdown of cells once removed from the body) and other forms of degradation.

While many histochemical treatments are capable of demonstrating broad categories of biochemicals in tissue sections (eg. proteins, glycoproteins, mucopolysaccharides, etc), most cannot distinguish between specific biomolecules. To do this a much more specific method must be used.

Immunohistochemistry is similar to immunofluorescence (see here) in that it uses antibodies to target and label specific proteins in tissue. Like immunofluorescence, immunohistochemistry uses a primary antibody directed against the target protein, and a labeled secondary antibody targeted against antibodies from the species in which the primary antibody was generated.

Immunohistochemistry differs from immunofluorescence in that in place of a fluorescent dye or fluorophore directly conjugated to the secondary antibody, immunohistochemistry uses a secondary antibody to which an enzyme has been conjugated. This enzyme (eg. horseradish peroxidase, or HRP) catalyses a change in a colourless compound (a chromogen) to produce a coloured precipitate at the place in the tissue which the antibody / enzyme complex is located.

The benefits of using immunohistochemistry are that the results can be demonstrated using routine brightfield microscopy (rather than a fluorescence microscope) allowing multiple channels to be viewed simultaneously, and that the coloured precipitate lasts longer in sections than the fluorophores do.

The signal produced by immunohistochemistry can be increased by using Avidin-Biotin conjugates (ABC). This increases the number of binding sites between the enzyme and the secondary antibody and therefore increases the signal strength.

Avidin is a protein derived from egg white which binds very strongly to the molecule biotin (also known as Vitamin B7). Due to its small size, biotin can be conjugated to large molecules like antibodies multiple times without affecting the function of the protein.

If ABCs are used, a biotinylated secondary antibody is bound to the primary antibody. If HRP conjugated to avidin is added to this, the avidin-HRP conjugate binds to the secondary antibody by way of the biotin molecules.