Immunohistochemical Assay Optimization – Tackling Intrinsic Study Variables


Immunohistochemistry (IHC) is employed routinely for investigative and diagnostic evaluations of antigen-antibody interactions within cells or tissues. While most IHC target antigens are proteins, other antigenic biological substrates can also be recognized by antibodies (e.g., carbohydrates, lipids, or nucleic acids). Accurate and reproducible results are achieved when procedures used to identify and localize tissue antigens are customized for a given study. EPL has extensive experience in providing IHC services to clients.  Our staff has a wealth of expertise and is well-equipped to develop and optimize study-specific IHC procedures as required. EPL staff routinely reviews the latest literature for information that allows us to develop the most current, expedient, and cost-effective IHC procedures, and works closely with antibody manufacturers and clients to ensure that study objectives are understood and production goals are met in a timely manner.

Due to the inherent complexity of many nonclinical research studies, variability in preanalytical and analytical methodology can present unique challenges for standardizing IHC methods that are effective or successful across studies, species, and tissue types. Preanalytical variables may directly influence the selection and effectiveness of analytical IHC techniques. Such variables include procedures performed to collect, prepare, and process histologic tissue specimens, examples of which are listed in Table 1.

Table 1. Preanalytical Variables That Can Affect IHC Methodology
Specimen Preparation Histologic Processing
Timing of tissue collection to prevent –the risk of autolytic tissue degradation Processing technique (e.g., frozen versus paraffin-embedded)
Fixation technique (e.g., perfusion versus immersion fixation) Storage conditions of embedded tissue blocks
Type of fixative and duration of fixation Slide type (e.g., chemical coated vs. positive charge slides)
Decalcification reagent and method Thickness of sections obtained at microtomy
Specimen type and size Unstained slide preparation, drying, and storage of sections


In each study, specimen preparation and histologic processing techniques should be controlled for consistency to ensure accuracy and reproducibility of the IHC method. For example, various solutions and fixation methods can be utilized to fix and preserve tissues.  Common fixative types include 10% neutral buffered formalin, 4% paraformaldehyde, and Bouin’s, Davidson’s, and glutaraldehyde solutions. Compared to simple tissue immersion, perfusion fixation often provides superior preservation.  This may be crucial for the evaluation of certain tissue types (e.g., brain), although in other instances the disadvantages of perfusion (e.g., increased time and cost) may outweigh the benefits. Experience has demonstrated that no single fixative or fixation protocol is ideal for all IHC assays. If antigen retrieval (AR) is deemed necessary, determination of the most appropriate AR technique will be based on multiple factors, such as morphologic and epitope stability in heat vs. enzymatic induced AR, the duration of fixation (e.g., the likelihood of over- or under-fixation), and the potential reversibility of aldehyde cross-linkages.

Successful performance of an IHC method relies on several analytical techniques that are optimized to promote specific antibody-antigen interactions in the target tissue.  Variables, such as reagent composition and reaction temperature/duration, are common analytical techniques that are optimized for each study (Table 2).

Table 2. IHC Procedures That May Require Optimization
Pretreatment Techniques Primary Antibody Specificity Detection & Visualization Systems Ancillary Techniques
Deparaffinization (for FFPEa sections) Reputable source (i.e., reported literature usage and vendor) Secondary (e.g., HRPb antibody vs. polymer IHC platforms: Manual (on-slide, free-floating) or automated (on-slide)
Antigen retrieval (e.g., enzymatic vs. heat induced) Clonality (monoclonal vs. polyclonal) Signal Amplification of secondary antibody – (e.g., avidin-biotin method) Washes: buffer composition, frequency, duration
Permeabilization: solvent or detergent based reagents Homology of immunogen to target antigen Detection: chromogenic or epifluorescent substrates, multiplex contrasting Controls: positive & negative tissue types, antibody isotype
Non-specific blocking (e.g., peroxidase, endogenous enzymes) Antibody concentration in diluent Counterstain: contrasting of substrates Mounting medium: aqueous, non-aqueous

a FFPE = Formalin fixed paraffin embedded

b HRP = Horseradish peroxidase


Antibodies produced for clinical IHC diagnostic use are manufactured in accordance with FDA regulations (21 CFR Part 864). Contingent on the test system, clinical IHC antibodies and reagents are labeled as either In Vitro Diagnostics (IVD) or Analyte Specific Reagents (ASR) to indicate the classification of regulatory production. However, IHC methods for nonclinical laboratory animal tissue routinely require antibodies and reagents that are classified as Research Use Only (RUO). The production of RUO antibodies does not require adherence to stringent regulatory requirements, and the binding affinity under particular preanalytical and analytical conditions is not always reported by the manufacturer nor demonstrated in published studies. Therefore, it is essential that RUO antibody performance is assessed prior to use in IHC studies. The antibody performance evaluation should include an antibody dilution series to identify the optimal concentration for antigen-antibody interaction that produces the highest signal-to-noise ratio. A performance assessment should additionally be conducted for each new manufactured lot of primary antibody to account for variability in inter-lot antibody performance.

The goal of IHC optimization is to develop a reproducible method that produces a clear signal with little or no background staining. Principles of process standardization, quality control, and tissue accountability that are routine histological practices are also applicable to IHC; however, IHC assays are additionally susceptible to problems of non-specific staining, antigen loss or masking, undesirable endogenous cross-reactivity, and low antibody-antigen homology. Polyclonal antibodies contain multiple clones of antibodies that are produced to target various epitopes on a given antigen and therefore, have higher levels of binding to a single antigen. Due to the high recognition of multiple epitopes, polyclonal antibodies can help amplify the signals of proteins with low expression.  Relative to polyclonal antibodies, monoclonal antibodies are highly specific and greatly reduce or eliminate cross-reactivity problems. However, antigen detection can still be hindered by the loss of epitope from tissue processing and IHC pretreatment. Blocking reagents (usually chemical- or protein-based reagents that bind non-specifically to the tissue but do not bind to the secondary antibody) have also been used to minimize the potential for false positive results. Because of these common pitfalls and variables, IHC methods must include appropriate positive and negative controls to assure confidence in the data.  The traditional approach for creating a negative control was to simply perform the IHC procedure with the primary antibody omitted.  This approach is no longer viewed favorably because it would fail to detect non-specific binding of the primary antibody to non-target antigens. A more appropriate method is to use isotypes derived from the primary antibody host species in place of the primary antibody to assess the specificity of the antibody-antigen interaction. Tissues known to contain the antigen of interest are routinely used as positive controls.  Such positive controls may be internal within the specimen of interest, -included as separate tissues, or both.  It may be necessary to use other types of controls to detect and guard against false positive results, e.g., omission of both the primary antibody and isotype control to pinpoint the source of noise or non-specific cross-reactivity, or controls used to ensure that the tissue does not contain endogenous markers (e.g., peroxidase or biotin).

All IHC optimization procedures are documented and follow established proprietary Standard Operating Procedures (SOPs), and are additionally conducted in accordance with Good Laboratory Practice (GLP) guidelines unless otherwise specified.  We welcome the opportunity to work with you to develop or optimize an efficacious IHC method designed specifically for your study.

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