Immunostaining - Methodological Overview

Methodological Overview

In immunostaining methods, an antibody is used to detect a specific protein epitope. These antibodies can be monoclonal or polyclonal. Detection of this first or primary antibody can be accomplished in multiple ways.

• The primary antibody can be directly labeled using an enzyme or fluorophore.
• The primary antibody can be labeled using a small molecule which interacts with a high affinity binding partner that can be linked to an enzyme or fluorophore. The biotin-streptavidin is one commonly used high affinity interaction.
• The primary antibody can be probed for using a broader species-specific secondary antibody that is labeled using an enzyme, or fluorophore.
• In the case of electron microscopy, antibodies are linked to a heavy metal atom.

As previously described, enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Fluorescent molecules can be visualised using fluorescence microscopy or confocal microscopy.