Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images

Abstract We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analys...

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Autores principales:	Roko Duplancic, Darko Kero
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c9cd6a6e8e1f471a89bf5dcbd1175b72
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spelling	oai:doaj.org-article:c9cd6a6e8e1f471a89bf5dcbd1175b722021-12-02T13:39:34ZNovel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images10.1038/s41598-021-88101-12045-2322https://doaj.org/article/c9cd6a6e8e1f471a89bf5dcbd1175b722021-04-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-88101-1https://doaj.org/toc/2045-2322Abstract We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.Roko DuplancicDarko KeroNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-17 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Roko Duplancic Darko Kero Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
description	Abstract We describe a novel approach for quantification and colocalization of immunofluorescence (IF) signals of multiple markers on high-resolution panoramic images of serial histological sections utilizing standard staining techniques and readily available software for image processing and analysis. Human gingiva samples stained with primary antibodies against the common leukocyte antigen CD45 and factors related to heparan sulfate glycosaminoglycans (HS GAG) were used. Expression domains and spatial gradients of IF signals were quantified by histograms and 2D plot profiles, respectively. The importance of histomorphometric profiling of tissue samples and IF signal thresholding is elaborated. This approach to quantification of IF staining utilizes pixel (px) counts and comparison of px grey value (GV) or luminance. No cell counting is applied either to determine the cellular content of a given histological section nor the number of cells positive to the primary antibody of interest. There is no selection of multiple Regions-Of-Interest (ROIs) since the entire histological section is quantified. Although the standard IF staining protocol is applied, the data output enables colocalization of multiple markers (up to 30) from a given histological sample. This can serve as an alternative for colocalization of IF staining of multiple primary antibodies based on repeating cycles of staining of the same histological section since those techniques require non standard staining protocols and sophisticated equipment that can be out of reach for small laboratories in academic settings. Combined with the data from ontological bases, this approach to quantification of IF enables creation of in silico virtual disease models.
format	article
author	Roko Duplancic Darko Kero
author_facet	Roko Duplancic Darko Kero
author_sort	Roko Duplancic
title	Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
title_short	Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
title_full	Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
title_fullStr	Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
title_full_unstemmed	Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images
title_sort	novel approach for quantification of multiple immunofluorescent signals using histograms and 2d plot profiling of whole-section panoramic images
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/c9cd6a6e8e1f471a89bf5dcbd1175b72
work_keys_str_mv	AT rokoduplancic novelapproachforquantificationofmultipleimmunofluorescentsignalsusinghistogramsand2dplotprofilingofwholesectionpanoramicimages AT darkokero novelapproachforquantificationofmultipleimmunofluorescentsignalsusinghistogramsand2dplotprofilingofwholesectionpanoramicimages
_version_	1718392642933358592

Novel approach for quantification of multiple immunofluorescent signals using histograms and 2D plot profiling of whole-section panoramic images

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