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A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry
I M S Paulsen1, H Dimke, S Frische
1University of Aarhus. imsp@biomed.au.dk.
Insights
Heat-induced epitope retrieval (HIER) can eliminate the need for paraffin removal in immunohistochemistry. This streamlined HIER procedure simplifies tissue preparation for formalin-fixed, paraffin-embedded samples.
Area of Science:
- Biomedical Sciences
- Histology
- Immunohistochemistry
Background:
- Heat-induced epitope retrieval (HIER) is a standard technique for immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissues.
- HIER protocols often include a dewaxing step using xylene, which involves heating paraffin sections above their melting point.
Purpose of the Study:
- To investigate whether the traditional xylene-based paraffin removal step is necessary before HIER.
- To evaluate the impact of omitting dewaxing on immunohistochemical staining results.
Main Methods:
- HIER was performed on FFPE tissue sections using vigorous boiling in a Tris/EGTA buffer (pH 9).
- Sections were analyzed with and without prior dewaxing in xylene.
- Immunohistochemical staining was assessed using 7 primary antibodies targeting various cellular locations.
- Staining intensity was evaluated through semiquantitative analysis and quantitative fluorescent secondary antibody-based assays.
- Nuclear staining with ToPro3 was also assessed.
Main Results:
- Omitting the prior dewaxing step in xylene had no discernible effect on staining patterns.
- Semiquantitative and quantitative analyses showed no consistent influence of omitting dewaxing on labeling intensity.
- Nuclear staining intensity remained unaffected by the omission of dewaxing.
Conclusions:
- The described HIER procedure effectively combines dewaxing, hydration, and epitope retrieval in a single step.
- This simplified protocol is suitable for immunohistochemistry on FFPE tissues, potentially streamlining workflow.
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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