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


RESULTS
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 degrees C or in the longer term at -20 degrees 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.


CONCLUSIONS
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.
Robertson
David
D
Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. david.robertson@icr.ac.uk
Savage
Kay
K
Reis-Filho
Jorge S
JS
Isacke
Clare M
CM
D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D015820:Cadherins / Q000378:metabolism; D024141:Collagen Type IV / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005557:Formaldehyde / Q000378:metabolism*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016612:Paraffin Embedding / Q000379:methods*; D011960:Receptors, Estrogen / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods*; D014746:Vimentin / Q000378:metabolism
D015820:Cadherins; D024141:Collagen Type IV; D011960:Receptors, Estrogen; D014746:Vimentin; D005557:Formaldehyde; D007633:Keratins