- Overview of Immunohistochemistry (includes links to individual pages on all aspects and stages of IHC)
Example of IHC Counterstaining
Hematoxylin is one of the most common dyes used in diagnostic histology, as well as a common nuclear counterstain in IHC. Oxidized hematoxylin is combined with aluminum ions to form an active metal-dye complex that stains the nuclei of mammalian cells blue by binding to lysine residues on nuclear histones, as opposed to other nuclear dyes that target the nucleic acids.
Nuclear fast red, also called Kernechtrot dye, is also a nuclear stain. Differences between this dye and hematoxylin, though, are that Nuclear fast red dyes nucleic acids, results in red nuclear staining and only takes 5 minutes to stain, instead of an hour with hematoxylin. Methyl green is also a nucleic acid dye that rapidly stains the nuclei green.
DAPI (4', 6-diamidino-2-phenylindole) and Hoechst are common nuclear dyes used for fluorescent IHC because they intercalate into the DNA to give a strong blue color under UV excitation. Propidium iodide is another nucleic acid dye that is frequently used to dye the nucleus red.
Phalloidin is a fungal toxin that binds to filamentous, but not globular (free) actin. The protein is commonly conjugated to fluorophores and used in fluorescent microscopic applications to label the actin cytoskeleton in cells. The color used is dependent upon the fluorophore that is conjugated, and a wide array of fluorophore-conjugated phalloidin products are commercially available.
Summary Table of Counterstains
|Chromogenic||Hematoxylin||Nuclei||Blue to violet|
|Chromogenic||Nuclear fast red (Kernechtrot)||Nucleic acids||Red|
|Chromogenic||Methyl green||Nucleic acids||Green|
|Fluorescent||Hoechst stain||Nucleic acids||Blue|
|Fluorescent||4', 6-diamidino-2-phenylindole (DAPI)||Nucleic acids||Blue|
|Fluorescent||Propidium iodide||Nucleic acids||Red|
|Fluorescent||Fluorophore-tagged phalloidin||Filamentous actin||Fluorophore-specific|
For Research Use Only. Not for use in diagnostic procedures.