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Related Concept Videos

Immunogold Electron Microscopy01:20

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Two basic types of preparation are used to visualize specimens with a light microscope: wet mounts and fixed specimens.
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Related Experiment Video

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Antigen Masking During Fixation and Embedding, Dissected.

Carla Rossana Scalia1, Giovanna Boi1, Maddalena Maria Bolognesi1

  • 1Dipartimento di Medicina e Chirurgia, Universitá degli Studi di Milano-Bicocca, Monza, Italy (CRS, GB, MMB, MM, FMB, SR, BEL, GC).

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
|November 1, 2016
PubMed
Summary
This summary is machine-generated.

Tissue processing steps like fixation and paraffin embedding cause antigen masking, reducing antibody detection. Antigen retrieval (AR) can reverse this masking, but also damages some epitopes. Frozen sections help study these effects.

Keywords:
FFPEantigen retrievalfixationformalinimmunostaining

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Area of Science:

  • Histopathology
  • Immunohistochemistry
  • Biochemistry

Background:

  • Antigen masking in routinely processed tissues is a complex phenomenon impacting immunohistochemical (IHC) staining.
  • Understanding the specific contributions of each processing step is crucial for optimizing antigen detection.

Purpose of the Study:

  • To dissect the impact of individual tissue processing steps on antigenicity.
  • To utilize frozen sections as a model system to investigate antigen masking and antibody accessibility.

Main Methods:

  • Comparison of antigen masking across variable fixation times.
  • Assessment of antigen detection following graded alcohol series and clearing agents.
  • Evaluation of antigen retrieval (AR) efficacy in reversing masking and preserving epitopes.
  • Use of frozen sections as surrogates for routine tissue processing.

Main Results:

  • Variable fixation times showed equivalent antigen masking; longer fixation (>24 hr) generally improved detection after AR.
  • Alcohol treatment enhanced staining, suggesting improved antibody access, potentially mimicked by detergents.
  • Clearing agents and paraffin embedding induced a second masking phase, linked to water removal.
  • Antigen retrieval (AR) effectively reversed masking from fixation and paraffin embedding but also caused epitope destruction.

Conclusions:

  • Tissue processing steps significantly influence antigen masking and antibody detection in IHC.
  • Frozen sections serve as a valuable tool for elucidating fixation and processing requirements for specific antigens.
  • Optimizing AR is essential to balance epitope unmasking and preservation.