Immunohistochemistry (IHC) uses antibodies to detect the expression and localization of proteins or other antigens in tissue sections. Antibody-antigen interactions are detected chromogenically with enzyme substrates or fluorescently with fluorescent dyes. Although IHC is not as quantitative as Western blotting or ELISA, it can provide valuable information on protein localization in the context of intact tissue. Protein expression profiling is of great value to pathologists, used to aid diagnosis of solid tumors and cytology specimens, and has been a primary diagnostic tool for nearly half a century.

Figure 1. Flow chart illustrating the immunohistochemistry protocol, and sequence of primary antibodies. (Osman TA, et al.; 2013)

Target Antigen

Antigens are proteins within cells or on their surface. Pathologists look for the presence of specific antigens (hundreds of antigens have been found to have diagnostic value) to aid diagnosis. Typically, pathologists use a "panel" of multiple antigens to comprehensively classify a specific tumor.

Antibody

The first stage of IHC is the application of a primary antibody that specifically binds to the target antigen.

There are two main types of primary antibodies: polyclonal and monoclonal. Polyclonal antibodies have affinity to multiple epitopes on the target protein and may have a stronger signal, but are also more likely to cross-react and produce nonspecific signals.

Monoclonal antibodies only have affinity for one epitope, which is beneficial for producing clearer and more specific staining, but they are less sensitive or less intense than polyclonal antibodies and are less resistant to the loss of epitopes caused by chemical treatment than polyclonal antibodies.

Secondary Antibodies

Next, the secondary antibody is combined with the primary antibody. This is called indirect IHC. Unlike using directly labeled primary antibodies, using secondary antibodies and associated detection systems amplifies the signal because more than one secondary antibody molecule binds to a single primary antibody.

Detection Systems

Immunoassays rely on the specificity of the primary antibody for the target antigen. You can use directly labeled primary antibodies for detection (direct method), or you can use labeled secondary antibodies for detection (indirect method). Antibody visualization is typically accomplished using fluorescent labels (e.g., FITC, R-PE, Alexa-Fluor^®) or using enzymes that convert soluble substrates into insoluble chromogenic products (e.g., HRP, AP).

Reagent

For detection systems with enzyme-labeled secondary antibodies, the substrate forms an insoluble chromogenic precipitate that can be observed under a microscope. There are two commonly used chromogens: DAB (brown) or AP-Red (red). DAB is suitable for most applications as it provides strong permanent staining. AP -Red (or other red chromogens) is primarily used in skin sections because in this case the brown DAB may be masked by the pigment granules of brown melanin.

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It is sometimes possible to use DAB and AP-Red simultaneously on the same tissue section, allowing the pathologist to view both antigens on a single slide. This process is called double dyeing.

15 Steps to Improve IHC

This guide provides actionable suggestions and solutions to common problems for obtaining the best IHC results. We hope to provide standardized operating procedures for each step to help troubleshoot problems when you do not obtain satisfactory IHC results.

Step 1 - Use High Quality Slices

• Use thin, flat sections, which need to be fully dried on the slide. It is appropriate to use charged slides or APES-coated slides for immunohistochemistry.
• Uneven, loosely adhered tissue can lead to uneven staining and high background.

Step 2 - Ensure Optimal Fixation

• Use familiar and stable fixation conditions (fixative solution type, pH, temperature, time) to ensure optimal fixation results. Samples should be inspected before fixation to determine whether they require further fixation.
• If the fixation conditions are inconsistent and the tissue is under-fixed or over-fixed, inconsistent results will be obtained, and it will be very difficult to troubleshoot the problem.

Step 3 - Avoid tissue adhesion

• Avoid using proteinaceous adhesives (glue, starch or gelatin) in the slide bath, especially when using charged slides.
• Protein-based adhesives will adhere to the surface of the charged slide, causing the tissue to not spread evenly on the slide, resulting in uneven staining.

Step 4 - Avoid Uneven Reagent Concentrations

• Use reagents with uniform concentration and no concentration gradient.

Step 5 - Choose Antibodies Carefully

• When selecting a primary antibody, its sensitivity and specificity must be fully considered. The antibodies sold by many different companies actually come from the same manufacturer, and the sales company only repackages or repackages them. When selecting an antibody, be sure to pay attention to its clone number.

Step 6 - Read the Parameter Sheet

• Know your primary antibodies. Check that your reagent method is suitable for your antibody. It is necessary to recheck the parameter sheet when purchasing a new batch of antibodies.

Step 7 - Optimize Antigen Retrieval Method

• Select the appropriate antigen retrieval method based on the characteristics of the selected antibody, the characteristics of the stained tissue, and the tissue fixation method.

Step 8 - Consider Antibody Cross-Reactivity

• Pay attention to every possible cross-reaction problem.

Step 9 - Inactivation of Endogenous Peroxidase

• For peroxidase-based detection systems, endogenous peroxidase must be inactivated.
• Non-specific staining is common in red blood cells, granulocytes, monocytes and muscle tissue. This is caused by incomplete blocking of endogenous peroxidase.

Step 10 - Avoid Background Staining

• Always use appropriate protein blocking solution.
• Background staining is sometimes caused by incomplete blocking of proteins.

Step 11 - Use an Appropriate Detection System

• Choose a detection system with accurate and specific staining properties and appropriate sensitivity.

Step 12 - Standardized Cleaning Procedure

• Use standardized cleaning steps throughout the process (including: duration, cleaning fluid volume, agitation method) to ensure stable test results.
• The staining results of the same antibody are different every time, which may be due to the operator's insufficient standard in the cleaning steps.

Step 13 - Optimize Counterstaining

• Nuclear counterstain levels are carefully adjusted and standardized to avoid masking positive staining. The counterstain should provide optimal contrast between the chromogen and background tissue elements. Appropriate counterstains should be selected for the chromogen used.
• Nuclear counterstain is sometimes very strong. This may mask weak specific staining.

Step 14 - Use Appropriate Control Slides

• Use appropriate positive and negative quality control slides for each experiment to more accurately detect and verify experimental results. Internal positive and negative controls are equally important as an effective way to ensure the quality of immunohistochemistry experiments.

Step 15 - Evaluate Experiment Results Carefully

• Know what to look for and where to look when evaluating stained sections and controls.
• As long as the detection area is positive, the staining of this section is qualified.

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1. Osman TA, et al.; Successful triple immunoenzymatic method employing primary antibodies from same species and same immunoglobulin subclass. Eur J Histochem. 2013, 57(3):e22.

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