After immunohistochemical staining the target antigen, a second stain is often applied to provide contast that helps the primary stain stand out. Many of these stains show specificity for discrete cellular compartments or antigens, while others will stain the whole cell. Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design.

Learn more

Example of IHC Counterstaining

Immunohistochemical detection of vimentin in tonsil tissue. Vimentin was detected in fixed human tonsil tissue by incubating cells with an anti-vimentin antibody followed by Thermo Scientific DyLight 549-Conjugated Goat Anti-Mouse Secondary Antibody (red). Cell nuclei were counterstained with Hoechst stain (blue).

Chromogenic Counterstains

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.

Fluorescent Counterstains

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

Common counterstains and their targets
Type Dye Target Color
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