A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue

  • I.M.S. Paulsen University of Aarhus, Denmark.
  • H. Dimke University of Southern Denmark, Denmark.
  • S. Frische | sfri@biomed.au.dk University of Aarhus, Denmark.

Abstract

Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue.

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Author Biographies

I.M.S. Paulsen, University of Aarhus
Department of Biomedicine
H. Dimke, University of Southern Denmark
Department of Cardiovascular and Renal Research, Institute of Molecular Medicine
S. Frische, University of Aarhus
Department of Biomedicine
Published
2015-11-03
Section
Technical Notes
Supporting Agencies
Danish National Research Foundation, Novo Nordisk Foundation, Carlsberg Foundation, A.P. Møller Foundation, Aarhus University, DFF, Medical Sciences and "MEMBRANES".
Keywords:
Immunolabelling, antibodies, histology, protein localization, HIER.
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How to Cite
Paulsen, I., Dimke, H., & Frische, S. (2015). A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue. European Journal of Histochemistry, 59(4). https://doi.org/10.4081/ejh.2015.2532