What is a Counterstain?

A counterstain is a stain or dye that has a color contrasting to the first or principal stain. After staining a target of interest with a primary antibody (principal stain), the counterstain is then applied as a second chemical stain. Selecting a color that contrasts the color of the primary stain helps to visualize and identify details of the cell types, organelles, structures in the tissue section and the exact location of positive cells. When selecting a counterstain for an immunostaining experiment, it is important to ensure the counterstains are of different color with sufficiently different absorption/emission characteristics so that they are easily distinguishable. For enzyme/chromogen detection systems, single nuclear counterstain is commonly used. For immunofluorescent detection systems, it is common to use a nuclear counterstain and a cell membrane counterstain. Below, we review common chromogenic and fluorescent counterstains that are used in immunohistochemistry staining.

Counterstains for Enzyme/Chromogen Detection Systems

Hematoxylin or haematoxylin is among the most commonly used nuclear counterstains for enzyme/chromogen detection systems. It is a naturally occurring chemical compound that is extracted from the logwood tree Hematoxylon campechianum. After exposure to air, hematoxylin oxidizes from colorless to a reddish/brown hematin (oxidized haematoxylin) and becomes negatively charged. By itself, oxidized hematoxylin stains DNA poorly due to its anionic qualities. However, when combined with a mordant (fixative) that is usually a metal salt (iron, aluminum, tungsten, and/or lead), a positively charged dye-mordant complex is formed facilitating binding to negatively charged molecules in cells and tissues and producing a stain with a deep blue to purple color. The intensity of staining depends on the amount of time tissues are stained, the concentration of hematoxylin used, the mordant used in the formulation and the method of hematoxylin staining used. There are two modes of hematoxylin staining – regressive and progressive. If a regressive mode of hematoxylin staining is used, the tissue is overstained with excess regressive hematoxylin (Harris) and then decolorized by immersion in an acid solution such as 1% acid alcohol. This decolorizing step is known as differentiation and removes the nonspecific background staining. On the other hand, in a progressive hematoxylin stain, the tissue is incubated in progressive hematoxylin (Mayer’s, Gill I) until the desired nuclear staining is achieved before being blued. The color intensity can be controlled through varying time in hematoxylin. Progressive stains are easier for optimizing development which allows for compatibility with alcohol soluble enzyme/substrate such as those in HRP enzyme/AEC substrate systems. Once the desired staining is achieved, the stain is then “blued” with bluing reagent to convert reddish purple to a deep blue/purple. Bluing agents are typically alkaline with a pH range of 7.5 to 9.0 and are tap water, ammonia water, or lithium carbonate. At acid pH, hematoxylins stain the nuclei red. At alkaline pH, hematoxylins stain the nuclei blue. Enzo Life Sciences provides our end-users with our HIGHDEF® Hematoxylin which is a proprietary, ready-to-use hematoxylin solution with enhancers to be used for histological sections. Enzo’s hematoxylin against a leading competitor’s demonstrated clearer staining and less background (Figure 1).

Figure 1: Enzo’s HIGHDEF® Hematoxylin against competitor hematoxylin.

Counterstaining the cytoplasm may be necessary to add more tinctorial qualities to nuclear stains such as hematoxylin. Hematoxylin is commonly paired with eosin, an orange/pink xanthene dye that is acidic and negatively charged and binds to positively charged proteins in the cytoplasm and connective tissue. Paired with hematoxylin, it makes for a compatible cytoplasmic counterpart that helps to clarify both nuclear and cytoplasmic structures in tissue. The hematoxylin and eosin combination is the most common staining technique used in histology. Other common nuclear counterstains include methylene blue and methyl green. Both are dyes that target nucleic acids. Methylene blue is a cationic dye widely used as a blue stain and does not require a mordant. It can be used to provide a quick nuclear counterstain that will bind to tissue anions such as carboxylic acid, sulphuric acid and phosphoric acid groups. Furthermore, it can stain both the cytoplasm and the nucleus. To get maximal staining, it is best to work in alkaline conditions; you can use potassium hydroxide in the buffering solution to raise the pH and ensure even staining of proteins and nucleic acids in your whole tissue. On the other hand, methyl green is another dye that targets nucleic acids. This is a blue/green nuclear stain that is used to differentiate between DNA and RNA in tissues. When used as a methyl green pyronin, methyl green stains the DNA and pyronin stains RNA.

Counterstains for Fluorescent Immunostaining Systems

Common counterstaining options for fluorescent immunostaining involve the use of fluorescent chemicals such as DAPI or Hoechst. DAPI (4', 6-diamidino-2-phenylindole) and Hoechst 33342 are common nuclear dyes that work by intercalating into the DNA and producing a strong blue color under UV excitation while staining the nuclei. DAPI is less membrane permeable than Hoescht and is typically used to stain fixed cells. Hoescht being membrane permeable allows for the flexibility of live cell or fixed cell applications. Both nuclear dyes can be cytotoxic to cells depending on the concentration. Furthermore, excessive photoconversion of DAPI and Hoescht may sometimes lead to the production of artifacts in images. When selecting fluorescent counterstains, keep in mind the filter sets available with the microscope being used and the absorption/emission spectra of other reporters being used for your samples. DAPI demonstrates relatively minimal cytoplasmic staining. Both DAPI and Hoescht produce a blue color. Propidium iodide is another common fluorescent nuclear counterstain. It is a DNA intercalating dye that binds DNA base pairs and forms a bright red color which is most suitable for IHC experiments using green reporter labels for sharp contrast. Some mounting media already come with a counterstain in it. This is helpful as it cuts down some steps in the staining process and are optimized with the right concentration of counterstain. We provide various mounting solution for chromogenic and fluorescent staining with or without counterstains to allow the user to choose their preferred staining procedure: HIGHDEF IHC mount, HIGHDEF IHC fluoromount, SEEBRIGHT® mounting medium and SEEBRIGHT® mounting medium with DAPI.

Enzo Life Sciences offers a complete range of products for IHC staining ranging from antigen retrieval, chromogens, detection and mounting media, to prepare your slides. Please check out our complete range of IHC products. For more suggestions on immunostaining, please see our IHC E-book, 10 Tips for Successful IHC and our IHC-related application notes. For any further questions and concerns regarding any of our products, please reach out to our Technical Support team. We are here to assist you with your IHC solutions!

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