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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
PubMed

Insights

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.

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.

Keywords:
IHCantigen retrievalcontrolepitopeformaldehydeimmunohistochemistryimmunostainpeptide

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