The Impact of pH on Antigen Retrieval in Immunohistochemistry

Multiplex ImageImmunohistochemistry (IHC) is a common technique used in biology and medicine to visualize and study the distribution of specific proteins within tissues. One critical step in the IHC process is antigen retrieval, which involves treating tissue samples to expose antigens that may be masked by fixation. The pH level at which antigen retrieval is performed can have a significant impact on the success and reliability of the IHC assay. In this blog, we will explore how pH can alter antigen retrieval and why it is essential to understand and control this variable in IHC experiments.

Antigen Retrieval:

Antigens are specific proteins or molecules of interest that histologists want to detect within tissue samples. During tissue fixation, these antigens can become cross-linked, making them inaccessible to antibodies throughout the IHC process. Antigen retrieval methods aim to reverse these cross-links, exposing the antigens and enabling antibody binding.

pH and Antigen Retrieval:

The pH level at which antigen retrieval is performed is a crucial factor that influences the effectiveness of the process. This is because pH can impact the chemical reactions involved in breaking down cross-links and restoring antigenicity.

Acidic pH:

Acidic conditions (pH below 6) are commonly used in antigen retrieval. Low pH can break the formalin-induced cross-links, making antigens more accessible. However, if you perform antigen retrieval with a buffer that is too acidic (i.e., with a pH below the optimal range), several potential issues can arise:

  • Tissue Damage: Excessive acidity can damage tissue structures, leading to the loss of cellular morphology. This can make it challenging to interpret the results and identify specific cell types or structures within the tissue.
  • Epitope Destruction: Extremely acidic conditions can cause epitope destruction. Epitopes are the specific regions of antigens recognized by antibodies. Destroying epitopes can result in false-negative or unreliable immunohistochemistry (IHC) results, as the antibodies may not bind effectively.
  • Protein Denaturation: Proteins in the tissue may denature under highly acidic conditions. This can result in the loss of antigenicity and hinder the ability of antibodies to detect the target proteins.
  • Reduced Signal: In cases where antigen retrieval is performed at an excessively acidic pH, you may observe weaker or no staining due to the abovementioned problems. This can lead to misinterpretation of results or difficulties in obtaining meaningful data.

Alkaline pH:

Alkaline conditions (pH above 9) are used less frequently but can be effective for some antigens. Alkaline pH can disrupt the methylene bridges in formalin-fixed tissues, facilitating antigen retrieval. However, antigen retrieval with a buffer that is too basic (i.e., with a pH above the optimal range) can also lead to several issues that can affect the success of your immunohistochemistry (IHC) experiment:

  • Tissue Damage: Like excessively acidic conditions, excessively basic conditions can damage tissue structures. This can result in the loss of cellular morphology and make it challenging to interpret the results accurately.
  • Epitope Destruction: Extremely alkaline conditions can lead to epitope destruction, where the specific regions on antigens recognized by antibodies become altered or damaged. This can result in false-negative or unreliable IHC results because the antibodies may not effectively bind to the target proteins.
  • Protein Denaturation: Proteins within the tissue can denature under highly basic conditions, leading to a loss of antigenicity. This can hinder the ability of antibodies to detect the target proteins, resulting in reduced or absent staining.
  • Non-specific Binding: Excessively basic conditions may lead to non-specific antibody binding, where antibodies bind to unintended targets in the tissue. This can produce background staining and reduce the specificity of your IHC results.

Buffer Selection:

Choosing the right buffer solution is crucial for antigen retrieval. Standard buffers include citrate, EDTA, and Tris-based solutions. Each buffer has a specific pH range where it is most effective, and the choice should be guided by the target antigen and tissue type.


Optimizing the pH for antigen retrieval is essential to achieve reliable and reproducible IHC results. Researchers should perform pH titration experiments to identify the optimal pH for their specific antigen and tissue. Additionally, controlling factors such as temperature, method, and retrieval time are equally important.


The pH level at which antigen retrieval is performed plays a significant role in the success of immunohistochemistry experiments. Researchers must carefully select and optimize the pH conditions for their specific antigens and tissues to ensure accurate and reliable results. Understanding how pH can alter antigen retrieval is a fundamental aspect of IHC and contributes to advancing research in biology and medicine.

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