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.

Protein Blockers

One major source of non-specific signal comes from intermolecular forces that promote antibody binding to other molecules in the specimen. To circumvent this, researchers add protein-based blocking agents that physically occupy non-specific binding sites prior to primary antibody addition. Depending on the blocking solution, the proteins within prevent non-specific binding to hydrophobic protein side chains or Fc receptors in the tissue. In theory, any protein that does not specifically bind to the epitope of interest can block unwanted interactions.

One common protein block is normal sera collected from healthy animals. Sera contain antibodies that bind non-specifically to Fc receptors, blocking the primary antibodies from interacting. The sera should originate from the species in which the secondary antibody was raised rather than that of the primary antibody animal. This ensures that the secondary antibody does not bind to the block and increase background signal.

Researchers also use protein block solutions, such as bovine serum albumin (BSA) and non-fat dry milk. BSA and non-fat dry milk proteins physically occupy binding spots that tend to interact non-specifically with antibodies through electrostatic and ionic interactions. An antibody specific for a target antigen should not have a greater affinity for the non-specific binding sites than the protein blockers. One advantage of this method is that there is no need to consider the source of the proteins in relation to the primary or secondary antibodies.

Animal-Free Blocker^® is a universal blocking agent and diluent perfect for researchers trying to avoid animal-derived proteins. An alternative to sera, BSA, casein, and non-fat dry milk, this reagent can be used for many applications, including IHC, nucleic acid blotting, and protein blotting.

Enzyme Blockers

Chromogenic detection methods use an enzyme linked to a secondary antibody to bind primary antibodies bound to the target. The most popular IHC detection substrates interact with horseradish peroxidase (HRP)– and alkaline phosphatase (AP)–conjugated antibodies. However, these enzymes are already present within some tissues, where they react with the detection substrate and cause background signal. To quench enzyme activity, enzyme blocking agents must be added prior to the detection step. Researchers often perform this blocking step before primary antibody incubation.

Peroxidase is especially common in the kidney, liver, and tissues containing red blood cells. Researchers often quench endogenous peroxidase with hydrogen peroxide. By simply dropping the chemical diluted in water, phosphate-buffered saline, or methanol onto prepared slides, the resulting reaction inactivates the enzyme and converts hydrogen peroxide to oxygen and water. Aqueous hydrogen peroxide solutions may destroy the underlying tissue architecture if the tissue is peroxidase-rich. In this case, methanol would be the better solvent choice.

If the detection substrate reacts with alkaline phosphatase, researchers may block endogenous enzyme activity with levamisole solution. Rather than applying this block prior to primary antibody addition, levamisole solution should be added to the alkaline phosphatase substrate solution right before detection.

BLOXALL^® Endogenous Blocking Solution is more effective than other enzyme blocking methods. It inactivates both endogenous peroxidase and alkaline phosphatase through a brief 10-minute incubation step. Many researchers prefer BLOXALL because it comes ready-to-use. Additionally, it is compatible with formalin-fixed, paraffin-embedded tissue sections, frozen tissue sections, and cell preparations.