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Related Experiment Video

Updated: Jan 30, 2026

Synthetic Antigen Controls for Immunohistochemistry

Synthetic Antigen Controls for Immunohistochemistry

Published on: August 23, 2021

Selecting an Optimal Positive IHC Control for Verifying Antigen Retrieval.

Seshi R Sompuram¹, Kodela Vani¹, Anika K Schaedle¹

¹Medical Discovery Partners LLC, Boston, Massachusetts.

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society

|January 11, 2019

Summary

Selecting appropriate positive controls is crucial for verifying heat-induced epitope retrieval (HIER) in immunohistochemistry (IHC). Low or intermediate analyte concentration controls are best for confirming HIER efficacy, unlike high concentrations which can be insensitive.

Keywords:

IHC antigen retrieval control epitope formaldehyde immunohistochemistry immunostain peptide

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Area of Science:

* Immunohistochemistry
* Molecular Biology
* Pathology

Background:

* Positive controls in immunohistochemistry (IHC) aim to detect issues in immunostaining and heat-induced epitope retrieval (HIER).
* The specific features of a control essential for verifying HIER remain unclear.
* Formalin-fixed tissues may not always be suitable for verifying HIER, as some stain without it.

Purpose of the Study:

* To investigate the critical features of positive controls for verifying HIER in IHC.
* To understand the impact of formalin fixation on epitope masking and HIER verification.
* To determine the optimal analyte concentration in positive controls for effective HIER assessment.

Main Methods:

* Utilized formalin-fixed peptide epitopes as a model system for precise control definition.
* Analyzed variable epitope masking caused by formalin fixation based on primary antibody recognition.
* Investigated the role of amino acid reactivity with formaldehyde in epitope masking variability.

Main Results:

* Formalin fixation causes variable epitope masking, dependent on the specific epitope and primary antibody.
* Amino acid composition influences an epitope's susceptibility to formaldehyde-induced masking.
* Positive controls with low or intermediate analyte concentrations are more sensitive for verifying HIER than high concentrations.

Conclusions:

* Not all formalin-fixed tissues are adequate controls for HIER verification.
* Epitope masking by formalin fixation varies significantly.
* Selecting positive controls with appropriate analyte concentration is critical for validating HIER in IHC.