Cargando…
A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue
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-...
Autores principales: | , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
PAGEPress Publications, Pavia, Italy
2015
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698609/ https://www.ncbi.nlm.nih.gov/pubmed/26708177 http://dx.doi.org/10.4081/ejh.2015.2532 |
_version_ | 1782408056639848448 |
---|---|
author | Paulsen, I.M.S. Dimke, H. Frische, S. |
author_facet | Paulsen, I.M.S. Dimke, H. Frische, S. |
author_sort | Paulsen, I.M.S. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4698609 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | PAGEPress Publications, Pavia, Italy |
record_format | MEDLINE/PubMed |
spelling | pubmed-46986092016-01-15 A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue Paulsen, I.M.S. Dimke, H. Frische, S. Eur J Histochem Technical Note 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. PAGEPress Publications, Pavia, Italy 2015-11-03 /pmc/articles/PMC4698609/ /pubmed/26708177 http://dx.doi.org/10.4081/ejh.2015.2532 Text en ©Copyright I.M.S. Paulsen et al. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Technical Note Paulsen, I.M.S. Dimke, H. Frische, S. A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title | A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title_full | A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title_fullStr | A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title_full_unstemmed | A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title_short | A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue |
title_sort | single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (hier) for immunohistochemistry in formalin-fixed paraffin-embedded tissue |
topic | Technical Note |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698609/ https://www.ncbi.nlm.nih.gov/pubmed/26708177 http://dx.doi.org/10.4081/ejh.2015.2532 |
work_keys_str_mv | AT paulsenims asinglesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue AT dimkeh asinglesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue AT frisches asinglesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue AT paulsenims singlesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue AT dimkeh singlesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue AT frisches singlesimpleprocedurefordewaxinghydrationandheatinducedepitoperetrievalhierforimmunohistochemistryinformalinfixedparaffinembeddedtissue |