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Dial up the Contrast with Counterstaining

When scientists detect targets of interest with immunohistochemistry (IHC), antibody binding and substrate detection alone often produce a perplexing picture where the target lacks context. They need something to fill in the blanks, and that’s where counterstaining comes in. Think of it as a tour guide. Counterstaining IHC samples after enzymatic detection spatially orients the target within its surroundings, which can help guide researchers as they analyze their results. Counterstains bind cellular components to color the neighboring cellular structures, giving researchers a look at the surrounding tissue architecture, as well as a better understanding of the morphological and spatial context of the target.

Common Counterstain Choices

The most commonly used counterstains target nuclei in simple procedures. Hematoxylin stains chromatin within nuclei blue, which contrasts with dark-hued peroxidase and alkaline phosphatase substrate precipitates. There are various formulations of hematoxylin, including Mayer’s, Gill’s, Harris, and Ehrlich’s, that differ in how the compound is oxidized and the choice of mordant that sets the dye on the specimen. H & E staining, a common technique in histology, combines hematoxylin in combination with eosin stain, which colors the extracellular matrix and cytoplasm pink.

Some detection reagents produce blue or purple precipitates, making a hematoxylin stain incompatible. In this case, researchers can use methyl green, a light green stain that complements a wide range of enzyme substrates. However, methyl green requires heating for proper uptake to a specimen. Another alternative to hematoxylin is nuclear fast red, a pink stain that contrasts with blue- or brown-hued detection substrates. Applying nuclear fast red requires just one simple step and takes mere minutes.

Key Considerations for Using Counterstains

Researchers should carefully choose their counterstain based on compatibility with the detection substrate. Counterstains that deposit too much color or too similar a color to the detection substrate can obscure the target signal or surrounding tissue, causing false positive or negative results.

To control the final appearance, researchers use most counterstains in either a progressive manner, in which the stain incubation time controls the intensity of color, or a regressive manner, in which the user overstains the specimen and carefully removes excess stain with reagents, such as acetic acid, to achieve the desired color. The stain application method—dropping stain onto the slide or immersing slides fully into the stain in batches—can also affect the intensity and distribution of the counterstain. Batch staining ensures consistency if multiple slides are being stained at once.

Many aspects of the IHC protocol affect the final appearance of the specimen. For example, pH changes alter the color of hematoxylin, and alcohol solvents may affect the solubility or intensity of the primary stain. Also, the heat and acetone rinse steps of the methyl green staining protocol may dissolve or discolor certain substrates. Early in the IHC workflow, the thoroughness of the fixation step affects how well the specimen picks up the primary antibody and counterstain. Additionally, stains have trouble penetrating thick sections, but detergents such as Tween 20 can help with uptake. Finally, stains behave differently depending on the tissue or species type.

To navigate these challenges and ensure the best results, researchers should run controls on fresh tissue sections with fresh stain to see how well their chosen counterstain works. Ideally, researchers will achieve a light and even counterstain before proceeding with IHC to identify the antigen targets.

To Counterstain, or Not to Counterstain?

Some researchers add counterstains to their IHC slides simply out of habit. However, counterstaining is unnecessary in some cases. Experiments using single antibodies typically benefit from a counterstain, but if there are multiple antibody targets on one slide, adding an additional color may detract from both the signals of interest and the results. Researchers should ask themselves if they will gain additional information from a counterstain, or if the extra color will obscure their data.

Counterstains are very useful for accentuating the primary signal and contributing additional information about the target. However, researchers should carefully consider whether they need a counterstain and which formula is best for their sample. Taking the time to choose the proper counterstain can make for a much more effective IHC stain.

Explore more counterstaining tips and caveats in IHC in this on-demand webinar.