How to Use Immunohistochemistry

How to Use Immunohistochemistry

Introduction of Immunohistochemistry

Immunohistochemistry (IHC) is a powerful technique widely employed in biological and medical research to visualize and study the distribution of proteins in tissue samples. This method allows researchers to gain valuable insights into the localization, abundance, and cellular expression patterns of specific proteins, contributing to a better understanding of various diseases and biological processes. In this article, we will provide a comprehensive guide on how to use immunohistochemistry effectively in your research endeavors.

The immunohistochemistry of endometrium tissue.Figure 1. The immunohistochemistry of endometrium tissue.


Immunohistochemistry involves the use of antibodies to detect and visualize specific proteins within tissue sections. The primary antibodies are designed to target particular antigens, and when combined with secondary antibodies labeled with detectable markers, they produce a visible signal. This method enables researchers to pinpoint the location of proteins of interest within cells or tissues.

Sample Preparation:

Before delving into the immunohistochemical process, proper sample preparation is crucial. Tissue fixation is the initial step, typically achieved using formalin. It preserves the tissue structure and prevents degradation of proteins. Following fixation, tissues are embedded in paraffin wax or frozen for sectioning. The choice between paraffin-embedded and frozen sections depends on the nature of the study and the antibodies used.

Antigen Retrieval:

Formalin fixation can lead to cross-linking of proteins, making them less accessible to antibodies. Antigen retrieval is a critical step to restore antigenicity by breaking these cross-links. Heat-induced epitope retrieval (HIER) is a common method, involving exposing tissue sections to high temperatures in a retrieval solution. This process enhances antibody binding by unfolding the proteins.

Blocking and Permeabilization:

To minimize non-specific binding of antibodies and enhance specificity, blocking agents are applied. Common blockers include serum proteins or bovine serum albumin (BSA). Additionally, permeabilization may be necessary for antibodies to access intracellular targets. Triton X-100 or Tween-20 are often used for this purpose.

Primary Antibody Incubation:

The primary antibody is the key player in immunohistochemistry. It binds specifically to the target protein within the tissue. The choice of the primary antibody depends on the protein of interest and the experimental design. Incubation times and temperatures vary but typically range from 30 minutes to overnight at 4°C.

Secondary Antibody Incubation:

Following primary antibody incubation, a secondary antibody labeled with a detectable marker is applied. Common markers include fluorophores or enzymes that produce a color reaction. This step amplifies the signal, increasing the sensitivity of the assay. Care must be taken to choose an appropriate secondary antibody that matches the host species of the primary antibody.


Visualization methods depend on the type of secondary antibody used. Fluorescent dyes allow for visualization under a fluorescence microscope, enabling multiple protein targets to be studied simultaneously. Enzyme-labeled secondary antibodies, such as horseradish peroxidase (HRP) or alkaline phosphatase, produce color reactions visible under a light microscope.


To enhance contrast and visualize tissue morphology, counterstaining with dyes such as hematoxylin is often performed. This step aids in the interpretation of immunohistochemical results by highlighting cellular structures.

Image Analysis:

Modern immunohistochemistry often involves digital image analysis, where software is employed to quantify staining intensity, distribution, and co-localization. This objective approach enhances the accuracy and reproducibility of results.


Immunohistochemistry is a delicate technique, and researchers may encounter challenges along the way. Common issues include high background staining, weak signals, or non-specific binding. Dilution optimization, changing blocking agents, or adjusting incubation times can often address these problems.


In conclusion, immunohistochemistry is a versatile and indispensable tool in biological and medical research. By following a systematic approach, researchers can unlock a wealth of information about protein expression and distribution within tissues. As technology continues to advance, immunohistochemistry remains a cornerstone in the pursuit of knowledge, providing valuable insights that contribute to our understanding of various diseases and biological processes.

  1. Sukswai N, Khoury JD. Immunohistochemistry Innovations for Diagnosis and Tissue-Based Biomarker Detection. Curr Hematol Malig Rep. 2019, 14(5):368-375.

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