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Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolab...

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Autores principales: Piña, Ricardo, Santos-Díaz, Alma I., Orta-Salazar, Erika, Aguilar-Vazquez, Azucena Ruth, Mantellero, Carola A., Acosta-Galeana, Isabel, Estrada-Mondragon, Argel, Prior-Gonzalez, Mara, Martinez-Cruz, Jadir Isai, Rosas-Arellano, Abraham
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836139/
https://www.ncbi.nlm.nih.gov/pubmed/35163349
http://dx.doi.org/10.3390/ijms23031426
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author Piña, Ricardo
Santos-Díaz, Alma I.
Orta-Salazar, Erika
Aguilar-Vazquez, Azucena Ruth
Mantellero, Carola A.
Acosta-Galeana, Isabel
Estrada-Mondragon, Argel
Prior-Gonzalez, Mara
Martinez-Cruz, Jadir Isai
Rosas-Arellano, Abraham
author_facet Piña, Ricardo
Santos-Díaz, Alma I.
Orta-Salazar, Erika
Aguilar-Vazquez, Azucena Ruth
Mantellero, Carola A.
Acosta-Galeana, Isabel
Estrada-Mondragon, Argel
Prior-Gonzalez, Mara
Martinez-Cruz, Jadir Isai
Rosas-Arellano, Abraham
author_sort Piña, Ricardo
collection PubMed
description Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay.
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spelling pubmed-88361392022-02-12 Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence Piña, Ricardo Santos-Díaz, Alma I. Orta-Salazar, Erika Aguilar-Vazquez, Azucena Ruth Mantellero, Carola A. Acosta-Galeana, Isabel Estrada-Mondragon, Argel Prior-Gonzalez, Mara Martinez-Cruz, Jadir Isai Rosas-Arellano, Abraham Int J Mol Sci Review Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay. MDPI 2022-01-26 /pmc/articles/PMC8836139/ /pubmed/35163349 http://dx.doi.org/10.3390/ijms23031426 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Review
Piña, Ricardo
Santos-Díaz, Alma I.
Orta-Salazar, Erika
Aguilar-Vazquez, Azucena Ruth
Mantellero, Carola A.
Acosta-Galeana, Isabel
Estrada-Mondragon, Argel
Prior-Gonzalez, Mara
Martinez-Cruz, Jadir Isai
Rosas-Arellano, Abraham
Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title_full Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title_fullStr Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title_full_unstemmed Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title_short Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence
title_sort ten approaches that improve immunostaining: a review of the latest advances for the optimization of immunofluorescence
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836139/
https://www.ncbi.nlm.nih.gov/pubmed/35163349
http://dx.doi.org/10.3390/ijms23031426
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