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Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins

Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a h...

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Autores principales: Padra, János Tamás, Lindén, Sara K
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8881734/
https://www.ncbi.nlm.nih.gov/pubmed/34420054
http://dx.doi.org/10.1093/glycob/cwab091
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author Padra, János Tamás
Lindén, Sara K
author_facet Padra, János Tamás
Lindén, Sara K
author_sort Padra, János Tamás
collection PubMed
description Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography–tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon).
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spelling pubmed-88817342022-02-28 Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins Padra, János Tamás Lindén, Sara K Glycobiology Chemical Biology Sulfomucins are in some body locations and species a normal occurrence, whereas in other situations, are a sign of pathology. Sulfomucin content on histological sections and isolated material is frequently analyzed with Alcian blue staining at pH 1.0. However, since the stain detects the charge, a high density of other charged molecules, such as sialic acids, has potential to impede specificity. Here, we compared the outcome from four staining protocols with the level of sulfation determined by liquid chromatography–tandem mass spectrometric analysis on samples from various tissues with variable sulfation and sialylation levels. We found that a protocol we designed, including rinsing with MetOH and 0.5 M NaCl buffer at pH 1.0, eliminates the false positive staining of tissues outperforming commonly recommended solutions. In tissues with low-to-moderately sulfated mucins (e.g. human stomach and salmonid epithelia), this method enables accurate relative quantification (e.g. sulfate scoring comparisons between healthy and diseased tissues), whereas the range of the method is not suitable for comparisons between tissues with high sulfomucin content (e.g. pig stomach and colon). Oxford University Press 2021-08-18 /pmc/articles/PMC8881734/ /pubmed/34420054 http://dx.doi.org/10.1093/glycob/cwab091 Text en © The Author(s) 2021. Published by Oxford University Press. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Chemical Biology
Padra, János Tamás
Lindén, Sara K
Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title_full Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title_fullStr Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title_full_unstemmed Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title_short Optimization of Alcian blue pH 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
title_sort optimization of alcian blue ph 1.0 histo-staining protocols to match mass spectrometric quantification of sulfomucins and circumvent false positive results due to sialomucins
topic Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8881734/
https://www.ncbi.nlm.nih.gov/pubmed/34420054
http://dx.doi.org/10.1093/glycob/cwab091
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