Analysis of Immunohistochemistry (IHC) Technical Principles and Key Points of Standardized Operation

Basic Principles and Key Elements of Immunohistochemistry

Immunohistochemistry (IHC) is a technique that uses the principle of specific binding between antigens and antibodies to localize, qualitatively analyze, and semi-quantitatively analyze target proteins in tissue sections or cell samples. Its core steps include antigen retrieval, blocking of nonspecific binding, primary antibody incubation, secondary antibody signal amplification, and chromogenic detection. In routine paraffin sections, formalin fixation and paraffin embedding can easily mask antigenic epitopes; therefore, the method and duration of antigen retrieval have a considerable impact on the results. Laboratories generally adopt common methods such as heat-induced epitope retrieval or enzymatic digestion retrieval based on the antibody datasheet and sample type.

The specificity and dilution ratio of the primary antibody are key determinants of staining quality. Validated antibodies should be prioritized, and preliminary experiments should first be performed at the recommended concentration. Different tissue sources, fixation times, and section thicknesses may require adjustment of incubation conditions. The inclusion of negative and positive controls helps exclude false positives or false negatives and is an indispensable step in standardized procedures.

Considerations for Sample Pretreatment and Staining Procedures

Sample pretreatment includes tissue collection, fixation, dehydration, embedding, sectioning, and deparaffinization and rehydration. Delayed or inadequate fixation can lead to antigen diffusion or degradation; generally, fixation should be performed as soon as possible after excision, commonly using neutral buffered formalin. Section thickness is usually controlled at 3–5 μm; sections that are too thick can easily cause background staining, while sections that are too thin may reduce signal intensity.

During staining, endogenous peroxidase blocking, serum blocking, and antibody incubation temperature and time should remain stable. When using an automated staining platform, instruments must be calibrated regularly, and reagent lines and incubation chamber temperature should be checked; for manual operation, it is recommended to establish standard operating procedures (SOPs) and train personnel. The development time for chromogenic substrates such as DAB should be controlled under the microscope to avoid excessive background.

Interpretation of Results and Analysis of Common Problems

Interpretation of immunohistochemistry results requires integration with tissue morphology and attention to whether positive signal localization is correct, such as cell membrane, cytoplasm, or nucleus. Common nonspecific staining may arise from endogenous biotin, peroxidase, or hydrophobic interactions and can be reduced by appropriate blocking agents and antibody diluents. Weakly positive or negative results require investigation of whether antigen retrieval was adequate, whether the primary antibody has lost activity, and whether the detection system sensitivity is sufficient.

Standardized procedures can not only improve comparability between experiments but also facilitate subsequent digital analysis and data consistency in multicenter studies. Laboratories can refer to relevant domestic and international guidelines to establish a quality control system suitable for their own sample types.