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Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains
Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cu...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173732/ https://www.ncbi.nlm.nih.gov/pubmed/34082785 http://dx.doi.org/10.1186/s13041-021-00799-2 |
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author | Tao-Cheng, Jung-Hwa Crocker, Virginia Moreira, Sandra Lara Azzam, Rita |
author_facet | Tao-Cheng, Jung-Hwa Crocker, Virginia Moreira, Sandra Lara Azzam, Rita |
author_sort | Tao-Cheng, Jung-Hwa |
collection | PubMed |
description | Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13041-021-00799-2. |
format | Online Article Text |
id | pubmed-8173732 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-81737322021-06-03 Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains Tao-Cheng, Jung-Hwa Crocker, Virginia Moreira, Sandra Lara Azzam, Rita Mol Brain Methodology Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13041-021-00799-2. BioMed Central 2021-06-03 /pmc/articles/PMC8173732/ /pubmed/34082785 http://dx.doi.org/10.1186/s13041-021-00799-2 Text en © The Author(s) 2021 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Methodology Tao-Cheng, Jung-Hwa Crocker, Virginia Moreira, Sandra Lara Azzam, Rita Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title | Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title_full | Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title_fullStr | Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title_full_unstemmed | Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title_short | Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
title_sort | optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8173732/ https://www.ncbi.nlm.nih.gov/pubmed/34082785 http://dx.doi.org/10.1186/s13041-021-00799-2 |
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