Multiple Immunofluorescence Labeling of Formalin-Fixed Paraffin-Embedded Tissue

  • David Robertson
  • Clare M. IsackeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 724)


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.

Key words

Immunofluorescent labeling Formalin-fixed paraffin-embedded FFPE Confocal microscopy Antigen retrieval Pathology 



This work was funded by Breakthrough Breast Cancer. We thank Jorge Reis-Filho, Kay Savage and Suzanne Parry for their help in developing this method.


  1. 1.
    Robertson, D., Savage, K., Reis-Filho, J. S. and Isacke, C. M. (2008) Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol. 9, 13.PubMedCrossRefGoogle Scholar
  2. 2.
    Simonavicius, N., Robertson, D., Bax, D. A., Jones, C., Huijbers, I. J. and Isacke, C. M. (2008) Endosialin (CD248) is a marker of tumor-associated pericytes in high-grade glioma. Mod Pathol. 21, 308–315.PubMedCrossRefGoogle Scholar
  3. 3.
    Pastrana, D. V., Tolstov, Y. L., Becker, J. C., Moore, P. S., Chang, Y. and Buck, C. B. (2009) Quantitation of human seroresponsiveness to Merkel cell polyomavirus. PLoS Pathog. 5, e1000578.PubMedCrossRefGoogle Scholar
  4. 4.
    Kendrick, H., Regan, J. L., Magnay, F. A., Grigoriadis, A., Mitsopoulos, C., Zvelebil, M., et al. (2008) Transcriptome analysis of mammary epithelial subpopulations identifies novel determinants of lineage commitment and cell fate. BMC Genomics 9, 591.PubMedCrossRefGoogle Scholar
  5. 5.
    Kosaka, N., Ogawa, M., Choyke, P. L., Karassina, N., Corona, C., McDougall, M., et al. (2009) In vivo stable tumor-specific painting in various colors using dehalogenase-based protein-tag fluorescent ligands. Bioconjug Chem. 20, 1367–1374.PubMedCrossRefGoogle Scholar
  6. 6.
    MacFadyen, J. R., Haworth, O., Roberston, D., Hardie, D., Webster, M. T., Morris, H. R., et al. (2005) Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium. FEBS Lett. 579, 2569–2575.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Breakthrough Centre, The Institute of Cancer ResearchRoyal Cancer HospitalLondonUK

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