Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue - PubMed (original) (raw)
Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue
David Robertson et al. BMC Cell Biol. 2008.
Abstract
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.
Conclusion: 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.
Figures
Figure 1
Expression of E-cadherin, Ksp-cadherin and collagen IV in kidney. 3 μm FFPE sections of adult human kidney were dewaxed, rehydrated and subject to antigen retrieval. A. For immunoperoxidase labelling, sections were incubated with the indicated antibodies and detection was achieved with the Vectastain avidin-biotin complex (ABC) system used according to the manufacturer's protocol (Vector Laboratories). Slides were counterstained with haematoxylin. B. Sections were stained as described in the Methods with anti-E-cadherin mAb and anti-Ksp-cadherin mAb or with anti-E-cadherin mAb and anti-collagen IV Ab followed by Alexa488-conjugated goat anti-mouse IgG2A and Alexa555 goat anti-mouse IgG1 or Alexa488-conjugated goat anti-mouse IgG2A and Alexa555 goat anti-rabbit Ig. Nuclei were counterstained with DAPI. Arrows indicate tubules expressing both cadherins, arrowheads indicate tubules expressing neither cadherin, * indicates tubules expressing E-cadherin alone. Scale bar, 50 μm.
Figure 2
Expression of cytokeratin 8/18, oestrogen receptor and vimentin in normal human breast and breast cancer. 3 μm FFPE sections of human adult breast containing both normal tissue and columnar cell change, and an ER-positive breast cancer showing areas of DCIS and invasive ductal carcinoma were stained anti-Ck 8/18 mAb, anti-vimentin mAb and anti-ER Ab followed by Alexa488 goat anti-mouse IgG1, Alexa633 goat anti-mouse IgG2A and Alexa555 goat anti-rabbit Ig. Nuclei were counterstained with DAPI. In all images colours are as follows: Ck 8/18 (green), ER (red), vimentin (yellow), DAPI (blue). A. 4 colour images, arrowheads indicate Ck 8/18-negative myoepithelial cells. B. Ck 8/18 and ER, arrows indicate ER expression in luminal epithelial cells with high Ck 8/18 expression in the normal breast. C. Ck 8/18 and vimentin, arrows indicate vimentin expression in the myoepithelial cells of columnar cell change, arrowheads indicate vimentin-positive, Ck 8/18-negative cells within the DCIS. D. Ck 8/18, ER and vimentin. Scale bar, 50 μm.
Figure 3
Immunofluorescence labelling of tissue microarrays slides. A tissue microarray containing 0.6 mm cores from 245 invasive breast cancers was stained as described in Figure 2. Ck 8/18 (green), ER (red), vimentin (yellow), DAPI (blue). Images from cores of 4 invasive ductal carcinomas (IDC) and 2 invasive lobular carcinomas (ILC) are shown.
Figure 4
Storage of FFPE samples after immunofluorescence labelling. A. 3 μm FPPE section of columnar cell change human breast was stained as described in Figure 2. Images were collected directly after labelling (day 1) and again following 70 days storage of the stained slides at 4°C. B. 3 μm FFPE section of human kidney was stained with anti-collagen IV Ab (red) and mouse anti-alpha smooth muscle actin mAb (green) followed by Alexa555 goat anti-rabbit Ig and Alexa488-conjugated goat anti-mouse IgG2A. Nuclei were counterstained with DAPI (blue). Images were collected directly after labelling (day1) and again following 270 days storage of the stained slides at -20°C. Scale bar, 50 μm.
References
- Bossard C, Jarry A, Colombeix C, Bach-Ngohou K, Moreau A, Loussouarn D, Mosnier JF, Laboisse CL. Phosphohistone H3 labelling for histoprognostic grading of breast adenocarcinomas and computer-assisted determination of mitotic index. J Clin Pathol. 2006;59:706–710. doi: 10.1136/jcp.2005.030452. - DOI - PMC - PubMed
- Ferri GL, Gaudio RM, Castello IF, Berger P, Giro G. Quadruple immunofluorescence: a direct visualization method. J Histochem Cytochem. 1997;45:155–158. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources