Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue



Investigating the expression of candidate genes in tissue samples usually involves either immunohistochemical labelling of formalin-fixed paraffin-embedded (FFPE) sections or immunofluorescence labelling of cryosections. Although both of these methods provide essential data, both have important limitations as research tools. Consequently, there is a demand in the research community to be able to perform routine, high quality immunofluorescence labelling of FFPE tissues.


We present here a robust optimised method for high resolution immunofluorescence labelling of FFPE tissues, which involves the combination of antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy. We demonstrate the utility of this method with examples of immunofluorescence labelling of human kidney, human breast and a tissue microarray of invasive human breast cancers. Finally, we demonstrate that stained slides can be stored in the short term at 4°C or in the longer term at -20°C prior to images being collected. This approach has the potential to unlock a large in vivo database for immunofluorescence investigations and has the major advantages over immunohistochemistry in that it provides higher resolution imaging of antigen localization and the ability to label multiple antigens simultaneously.


This method provides a link between the cell biology and pathology communities. For the cell biologist, it will enable them to utilise the vast archive of pathology specimens to advance their in vitro data into in vivo samples, in particular archival material and tissue microarrays. For the pathologist, it will enable them to utilise multiple antibodies on a single section to characterise particular cell populations or to test multiple biomarkers in limited samples and define with greater accuracy cellular heterogeneity in tissue samples.

David Robertson, Kay Savage, Jorge S Reis-Filho and Clare M Isacke

Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK

BMC Cell Biology 2008, 9:13doi:10.1186/1471-2121-9-13

The electronic version of this article is the complete one and can be found online at:


Immunohistochemistry (IHC) is one of the pillars of modern diagnostic pathology and a fundamental research tool in both pathology and translational research laboratories. Currently, labelling of FFPE specimens most commonly involves a biotinylated secondary antibody followed by an avidin-biotin-peroxidase complex and development with a soluble chromogenic substrate. This approach is robust and reliable, and increasingly can be automated for labelling, image acquisition and scoring. However, as a research tool there are three major limitations. First, it is primarily used to reveal one protein at a time; multiple colour approaches by combining peroxidase with other development systems are less than satisfactory and cannot be used to examine the co-localization of two antigens in the same subcellular compartment. Second, the resolution of antigen localization is limited due to the chromogenic substrate precipitate and the thickness (3 – 4 μm) of the sections imaged in the light microscope. Third, chromogenic systems saturate easily, which restricts semi-quantitative analysis.

Immunofluorescence labelling, on the other hand, has the capability for multiple labelling and is of higher resolution due to the fluorophores being directly conjugated to the antibody. Nevertheless, immunofluorescence labelling is not often used for FFPE specimens, the perceived mantra being that the inherent autofluorescence of such specimens makes high quality immunofluorescence imaging capricious. This has placed two severe restrictions on investigators. First, it has limited fluorescence imaging to tissue cryosections and hence restricts analysis of clinical material. Second, as cell and tissue preservation is lower in cryosections than in FFPE sections, the quality of morphological findings is frequently compromised.

This is particularly the case in tissues that are difficult to cryosection, for example cartilage, bone and those that contain a high fat content such as the breast. In recent years, there have been a number of reports describing the immunofluorescence labelling of FFPE sections [1-10] but, for a variety of reasons, these methods have not been taken up widely by the scientific community (see Discussion). Similarly, quantitative immunofluorescence labelling of FFPE material, particularly that in tissue microarrays, has been achieved by the development of computer assisted fluorescence imaging systems [11].

Although these systems have an important role in translational research, there is still an urgent need for a high resolution method which can be employed by the wider research community. To this end, we have taken a systematic approach to develop a robust protocol for coupling antigen retrieval, indirect immunofluorescence and confocal laser scanning microscopy to image FFPE sections. Using this method, we demonstrate that multicolour immunofluorescence imaging of FFPE material is readily achievable and that this method provides excellent images. Of note, the data shown here were not subjected to any image manipulation.