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Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development

Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of c...

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Autores principales: Pérez Arévalo, Andrea, Lutz, Anne-Kathrin, Atanasova, Ekaterina, Boeckers, Tobias M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9560478/
https://www.ncbi.nlm.nih.gov/pubmed/36227942
http://dx.doi.org/10.1371/journal.pone.0275780
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author Pérez Arévalo, Andrea
Lutz, Anne-Kathrin
Atanasova, Ekaterina
Boeckers, Tobias M.
author_facet Pérez Arévalo, Andrea
Lutz, Anne-Kathrin
Atanasova, Ekaterina
Boeckers, Tobias M.
author_sort Pérez Arévalo, Andrea
collection PubMed
description Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages.
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spelling pubmed-95604782022-10-14 Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development Pérez Arévalo, Andrea Lutz, Anne-Kathrin Atanasova, Ekaterina Boeckers, Tobias M. PLoS One Lab Protocol Whole animal perfusion is a well-established method that has been used for the past decades in multiple research fields. Particularly, it has been very important for the study of the brain. The rapid and uniform fixation of tissue is essential for the preservation of its integrity and the study of complex structures. For small tissue pieces submerging in formaldehyde solution oftentimes is sufficient to get a good fixation, larger tissues or organs with a more complicated structure present a greater difficulty. Here, we report the precise parameters to successfully perform trans-cardiac perfusion of neonatal mouse pups that allows a uniform fixation of the whole body for subsequent structural analysis and immunohistochemistry. In comparison to standard perfusion procedures of adult mice, changes in the pump velocity, the buffer volume and in the needle size lead to high quality fixation of neonatal mice pups. Further, we present a whole-body section staining, which results in a highly specific immunofluorescence signal suited for detailed analysis of multiple tissues or systems at the same time. Thus, our protocol provides a reproducible and reliable method for neonatal perfusion and staining that can rapidly be applied in any laboratory. It allows a high quality analysis of cellular structures and expression profiles at early developmental stages. Public Library of Science 2022-10-13 /pmc/articles/PMC9560478/ /pubmed/36227942 http://dx.doi.org/10.1371/journal.pone.0275780 Text en © 2022 Pérez Arévalo et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Lab Protocol
Pérez Arévalo, Andrea
Lutz, Anne-Kathrin
Atanasova, Ekaterina
Boeckers, Tobias M.
Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title_full Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title_fullStr Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title_full_unstemmed Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title_short Trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
title_sort trans-cardiac perfusion of neonatal mice and immunofluorescence of the whole body as a method to study nervous system development
topic Lab Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9560478/
https://www.ncbi.nlm.nih.gov/pubmed/36227942
http://dx.doi.org/10.1371/journal.pone.0275780
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