Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.

The combined use of immunolabeling and confocal microscopy offers a robust tool for the comprehensive examination of red blood cells, allowing clear visualization of specific proteins and their roles in the cell. While immunolabeling is a widely employed technique, it presents specific challenges in...

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Autores: Rey-Barroso L, Roldán M, Frías M, Burgos-Fernández FJ, Isola I, Llobet AR, Sarrate E, Vilaseca M
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2025
País:España
Institución:Fundació Sant Joan de Déu
Repositorio:r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu
OAI Identifier:oai:fsjd.fundanetsuite.com:p29097
Acceso en línea:https://fsjd.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=29097
Access Level:acceso abierto
Palabra clave:Confocal microscopy
Flow cytometry
Hemoglobin F
Immunofluorescence
Red blood cells
Sickle cell anemia
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spelling Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.Rey-Barroso LRoldán MFrías MBurgos-Fernández FJIsola ILlobet ARSarrate EVilaseca MConfocal microscopyFlow cytometryHemoglobin FImmunofluorescenceRed blood cellsSickle cell anemiaThe combined use of immunolabeling and confocal microscopy offers a robust tool for the comprehensive examination of red blood cells, allowing clear visualization of specific proteins and their roles in the cell. While immunolabeling is a widely employed technique, it presents specific challenges in the study of red blood cells, including difficulties with membrane permeabilization, the appearance of background fluorescence, detection of very faint signals, interference from other blood components, and, notably, poor preservation resulting from an incorrect choice of fixation components. Adding to this, fresh blood samples are very labile and need to be preserved as soon as possible, ideally immediately after extraction. Addressing these challenges requires careful experimental design to ensure assay specificity and sensitivity, often complicated by incomplete or unclear information in the literature. In this study, we have compiled available bibliographic data regarding key aspects of red blood cell immunolabeling and devised a detailed immunostaining protocol to investigate the presence of Hemoglobin F in patients with sickle cell anemia treated with hydroxyurea. This protocol ensures proper preservation of red blood cell characteristics and may find application in immunolabeling with other intracellular antibodies.PERGAMON-ELSEVIER SCIENCE LTD2025info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://fsjd.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=29097MICRONISSN: 09684328ISSNe: 18784291reponame:r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déuinstname:Fundació Sant Joan de DéuInglésinfo:eu-repo/semantics/openAccessoai:fsjd.fundanetsuite.com:p290972026-05-27T12:37:41Z
dc.title.none.fl_str_mv Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
title Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
spellingShingle Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
Rey-Barroso L
Confocal microscopy
Flow cytometry
Hemoglobin F
Immunofluorescence
Red blood cells
Sickle cell anemia
title_short Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
title_full Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
title_fullStr Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
title_full_unstemmed Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
title_sort Optimized protocol for intracellular labeling of red blood cells with anti-hemoglobin F for confocal microscopy analysis.
dc.creator.none.fl_str_mv Rey-Barroso L
Roldán M
Frías M
Burgos-Fernández FJ
Isola I
Llobet AR
Sarrate E
Vilaseca M
author Rey-Barroso L
author_facet Rey-Barroso L
Roldán M
Frías M
Burgos-Fernández FJ
Isola I
Llobet AR
Sarrate E
Vilaseca M
author_role author
author2 Roldán M
Frías M
Burgos-Fernández FJ
Isola I
Llobet AR
Sarrate E
Vilaseca M
author2_role author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Confocal microscopy
Flow cytometry
Hemoglobin F
Immunofluorescence
Red blood cells
Sickle cell anemia
topic Confocal microscopy
Flow cytometry
Hemoglobin F
Immunofluorescence
Red blood cells
Sickle cell anemia
description The combined use of immunolabeling and confocal microscopy offers a robust tool for the comprehensive examination of red blood cells, allowing clear visualization of specific proteins and their roles in the cell. While immunolabeling is a widely employed technique, it presents specific challenges in the study of red blood cells, including difficulties with membrane permeabilization, the appearance of background fluorescence, detection of very faint signals, interference from other blood components, and, notably, poor preservation resulting from an incorrect choice of fixation components. Adding to this, fresh blood samples are very labile and need to be preserved as soon as possible, ideally immediately after extraction. Addressing these challenges requires careful experimental design to ensure assay specificity and sensitivity, often complicated by incomplete or unclear information in the literature. In this study, we have compiled available bibliographic data regarding key aspects of red blood cell immunolabeling and devised a detailed immunostaining protocol to investigate the presence of Hemoglobin F in patients with sickle cell anemia treated with hydroxyurea. This protocol ensures proper preservation of red blood cell characteristics and may find application in immunolabeling with other intracellular antibodies.
publishDate 2025
dc.date.none.fl_str_mv 2025
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://fsjd.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=29097
url https://fsjd.fundanetsuite.com/Publicaciones/ProdCientif/PublicacionFrw.aspx?id=29097
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv PERGAMON-ELSEVIER SCIENCE LTD
publisher.none.fl_str_mv PERGAMON-ELSEVIER SCIENCE LTD
dc.source.none.fl_str_mv MICRON
ISSN: 09684328
ISSNe: 18784291
reponame:r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu
instname:Fundació Sant Joan de Déu
instname_str Fundació Sant Joan de Déu
reponame_str r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu
collection r-FSJD. Repositorio Institucional de Producción Científica de la Fundació Sant Joan de Déu
repository.name.fl_str_mv
repository.mail.fl_str_mv
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