Cargando…
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...
Autores principales: | , , , , , , , , , |
---|---|
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 |
_version_ | 1784649603394568192 |
---|---|
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. |
format | Online Article Text |
id | pubmed-8836139 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT pinaricardo tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT santosdiazalmai tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT ortasalazarerika tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT aguilarvazquezazucenaruth tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT mantellerocarolaa tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT acostagaleanaisabel tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT estradamondragonargel tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT priorgonzalezmara tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT martinezcruzjadirisai tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence AT rosasarellanoabraham tenapproachesthatimproveimmunostainingareviewofthelatestadvancesfortheoptimizationofimmunofluorescence |