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Optimization of immunostaining on flat-mounted human corneas

PURPOSE: In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts freq...

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Autores principales: Forest, Fabien, Thuret, Gilles, Gain, Philippe, Dumollard, Jean-Marc, Peoc’h, Michel, Perrache, Chantal, He, Zhiguo
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Molecular Vision 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704774/
https://www.ncbi.nlm.nih.gov/pubmed/26788027
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author Forest, Fabien
Thuret, Gilles
Gain, Philippe
Dumollard, Jean-Marc
Peoc’h, Michel
Perrache, Chantal
He, Zhiguo
author_facet Forest, Fabien
Thuret, Gilles
Gain, Philippe
Dumollard, Jean-Marc
Peoc’h, Michel
Perrache, Chantal
He, Zhiguo
author_sort Forest, Fabien
collection PubMed
description PURPOSE: In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. METHODS: The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, −20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch’s endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. RESULTS: Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional −20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. CONCLUSIONS: Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia.
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spelling pubmed-47047742016-01-19 Optimization of immunostaining on flat-mounted human corneas Forest, Fabien Thuret, Gilles Gain, Philippe Dumollard, Jean-Marc Peoc’h, Michel Perrache, Chantal He, Zhiguo Mol Vis Research Article PURPOSE: In the literature, immunohistochemistry on cross sections is the main technique used to study protein expression in corneal endothelial cells (ECs), even though this method allows visualization of few ECs, without clear subcellular localization, and is subject to the staining artifacts frequently encountered at tissue borders. We previously proposed several protocols, using fixation in 0.5% paraformaldehyde (PFA) or in methanol, allowing immunostaining on flatmounted corneas for proteins of different cell compartments. In the present study, we further refined the technique by systematically assessing the effect of fixative temperature. Last, we used optimized protocols to further demonstrate the considerable advantages of immunostaining on flatmounted intact corneas: detection of rare cells in large fields of thousands of ECs and epithelial cells, and accurate subcellular localization of given proteins. METHODS: The staining of four ubiquitous proteins, ZO-1, hnRNP L, actin, and histone H3, with clearly different subcellular localizations, was analyzed in ECs of organ-cultured corneas. Whole intact human corneas were fixed for 30 min in 0.5% paraformaldehyde or pure methanol at four temperatures (4 °C for PFA, −20 °C for methanol, and 23, 37, and 50 °C for both). Experiments were performed in duplicate and repeated on three corneas. Standardized pictures were analyzed independently by two experts. Second, optimized immunostaining protocols were applied to fresh corneas for three applications: identification of rare cells that express KI67 in the endothelium of specimens with Fuch’s endothelial corneal dystrophy (FECD), the precise localization of neural cell adhesion molecules (NCAMs) in normal ECs and of the cytokeratin pair K3/12 and CD44 in normal epithelial cells, and the identification of cells that express S100b in the normal epithelium. RESULTS: Temperature strongly influenced immunostaining quality. There was no ubiquitous protocol, but nevertheless, room temperature may be recommended as first-line temperature during fixation, instead of the conventional −20 °C for methanol and 4 °C for PFA. Further optimization may be required for certain target proteins. Optimized protocols allowed description of two previously unknown findings: the presence of a few proliferating ECs in FECD specimens, suggesting ineffective compensatory mechanisms against premature EC death, and the localization of NCAMs exclusively in the lateral membranes of ECs, showing hexagonal organization at the apical pole and an irregular shape with increasing complexity toward the basal pole. Optimized protocols were also effective for the epithelium, allowing clear localization of cytokeratin 3/12 and CD44 in superficial and basal epithelial cells, respectively. Finally, S100b allowed identification of clusters of epithelial Langerhans cells near the limbus and more centrally. CONCLUSIONS: Fixative temperature is a crucial parameter in optimizing immunostaining on flatmounted intact corneas. Whole-tissue overview and precise subcellular staining are significant advantages over conventional immunohistochemistry (IHC) on cross sections. This technique, initially developed for the corneal endothelium, proved equally suitable for the corneal epithelium and could be used for other superficial mono- and multilayered epithelia. Molecular Vision 2015-12-30 /pmc/articles/PMC4704774/ /pubmed/26788027 Text en Copyright © 2015 Molecular Vision. http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited, used for non-commercial purposes, and is not altered or transformed.
spellingShingle Research Article
Forest, Fabien
Thuret, Gilles
Gain, Philippe
Dumollard, Jean-Marc
Peoc’h, Michel
Perrache, Chantal
He, Zhiguo
Optimization of immunostaining on flat-mounted human corneas
title Optimization of immunostaining on flat-mounted human corneas
title_full Optimization of immunostaining on flat-mounted human corneas
title_fullStr Optimization of immunostaining on flat-mounted human corneas
title_full_unstemmed Optimization of immunostaining on flat-mounted human corneas
title_short Optimization of immunostaining on flat-mounted human corneas
title_sort optimization of immunostaining on flat-mounted human corneas
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704774/
https://www.ncbi.nlm.nih.gov/pubmed/26788027
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