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Updated: Jun 4, 2026
Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens
Published on: January 21, 2019
Multiple immunofluorescence labeling of formalin-fixed paraffin-embedded tissue
David Robertson1, Clare M Isacke
1Breakthrough Centre, The Institute of Cancer Research, Royal Cancer Hospital, London, UK.
Abstract:
Multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissue is not a routinely used method. At least in part, this is due to the perception that the innate autofluorescence of the FFPE material forbids the use of immunofluorescent labeling. As a result, immunohistochemical (immunoperoxidase) staining of FFPE material or cryosectioning methods is used instead. In this chapter, we describe a robust optimized method for high-resolution immunofluorescence labeling of FFPE tissue that involves the combination of antigen retrieval, indirect immunofluorescence, and confocal laser scanning microscopy. Once such samples have been prepared and imaged by confocal microscopy, they can be stored at -20°C for extensive periods (>250 days) and reexamined with minimal loss of quality. As a consequence, this method has the potential to open up the large archival sample collections to multiple immunofluorescent investigations.
Insights
This study presents an optimized method for multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissues. This technique overcomes autofluorescence issues, enabling detailed analysis of archival tissue samples.
Area of Science:
- Histology
- Immunofluorescence
- Microscopy
Background:
- Multiple immunofluorescent labeling of FFPE tissue is not routine.
- Autofluorescence of FFPE tissue is a major limitation.
- Immunohistochemistry or cryosectioning are common alternatives.
Purpose of the Study:
- To describe an optimized method for high-resolution immunofluorescence labeling of FFPE tissue.
- To overcome FFPE autofluorescence for immunofluorescence applications.
- To enable multi-labeling investigations on archival FFPE samples.
Main Methods:
- Optimized protocol for FFPE tissue preparation.
- Combination of antigen retrieval and indirect immunofluorescence.
- Confocal laser scanning microscopy for imaging.
Main Results:
- Successful high-resolution immunofluorescence labeling of FFPE tissue achieved.
- Method overcomes inherent FFPE autofluorescence.
- Prepared samples are stable for long-term storage (>250 days) at -20°C with minimal quality loss.
Conclusions:
- This optimized method allows for robust multiple immunofluorescent labeling of FFPE tissues.
- The technique facilitates the use of large archival FFPE sample collections for immunofluorescence studies.
- This approach expands the utility of FFPE samples in biomedical research.
