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Cell Membrane Nanostructure is Altered by Heat-Induced Antigen Retrieval: A Possible Consequence for Immunocytochemical Detection of Membranous Antigens

Published online by Cambridge University Press:  14 November 2019

Katerina Cizkova ,
Jakub Malohlava  and
Zdenek Tauber
Katerina Cizkova
Affiliation:
Department of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University, 779 00Olomouc, Czech Republic Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 779 00Olomouc, Czech Republic
Jakub Malohlava
Affiliation:
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, 779 00Olomouc, Czech Republic Department of Medical Biophysics, Faculty of Medicine and Dentistry, Palacky University, 779 00Olomouc, Czech Republic
Zdenek Tauber*
Affiliation:
Department of Histology and Embryology, Faculty of Medicine and Dentistry, Palacky University, 779 00Olomouc, Czech Republic
*
*Author for correspondence: Zdenek Tauber, E-mail: zdenek.tauber@upol.cz
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Abstract

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

Keywords

atomic force microscopycell surface nanostructureimmunostainingmembrane antigens
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 26 , Issue 1 , February 2020 , pp. 139 - 147
Copyright
Copyright © Microscopy Society of America 2019

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