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Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections
G Cattoretti1, S Pileri, C Parravicini
1Department of Pathology, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy.
The Journal of Pathology
|October 1, 1993
Summary
Optimized antigen unmasking on formalin-fixed, paraffin-embedded tissues using enzymatic and non-enzymatic methods. Non-enzymatic treatments, like microwave boiling, offer a powerful alternative for improving antibody-based immunostaining.
Area of Science:
- Histopathology
- Immunohistochemistry
- Biochemistry
Background:
- Formalin-fixed, paraffin-embedded (FFPE) tissues are crucial for diagnostics.
- Antigen retrieval is essential for antibody binding in FFPE sections.
- Enzymatic methods are traditional but can be harsh.
Purpose of the Study:
- To optimize and compare enzymatic and non-enzymatic antigen unmasking methods.
- To evaluate the efficacy of different unmasking techniques across various antibodies.
- To elucidate the mechanism of non-enzymatic antigen unmasking.
Main Methods:
- Optimized non-enzymatic antigen unmasking using microwave boiling in salt solution or urea.
- Compared non-enzymatic methods with enzymatic digestion (trypsin, pronase).
- Tested unmasking treatments with a panel of 256 antibodies.
Main Results:
- Non-enzymatic methods (microwave boiling) effectively unmasked antigens.
- Both enzymatic and non-enzymatic methods are effective, with some antigens showing selective benefit from one over the other.
- Protein denaturation is the likely mechanism for microwave-based unmasking.
Conclusions:
- Non-enzymatic antigen unmasking is a powerful tool for FFPE immunostaining.
- These methods broaden the utility of antibodies in diagnostic pathology.
- Non-enzymatic methods should complement traditional enzymatic approaches.