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The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices

The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices
The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy (EM). However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles, and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts, and knowing how to avoid them, takes on special urgency. We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a “before-and-after” experiment. We applied super-resolution shadow imaging (SUSHI) to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters. While the ECS volume fraction (VF) and microanatomic organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted. Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphologic preservation of cells and tissues.

PFA fixation artefacts, STED microscopy, dendrites, extracellular space, micro anatomy, mouse brain tissue
2373-2822
Idziak, Agata
6737d346-ec15-4810-8ae3-a2f96e30db36
Inavalli, V V G Krishna
db7ab576-a272-4f04-b938-3d1772ffbd01
Bancelin, Stephane
fbcaac42-f761-4f45-8d59-aa4d132fa6ab
Arizono, Misa
141dead8-892c-46bf-ae8d-79eebd049de8
Nägerl, U Valentin
cbf97dc1-771a-43ae-b3c6-86f34040997b
Idziak, Agata
6737d346-ec15-4810-8ae3-a2f96e30db36
Inavalli, V V G Krishna
db7ab576-a272-4f04-b938-3d1772ffbd01
Bancelin, Stephane
fbcaac42-f761-4f45-8d59-aa4d132fa6ab
Arizono, Misa
141dead8-892c-46bf-ae8d-79eebd049de8
Nägerl, U Valentin
cbf97dc1-771a-43ae-b3c6-86f34040997b

Idziak, Agata, Inavalli, V V G Krishna, Bancelin, Stephane, Arizono, Misa and Nägerl, U Valentin (2023) The impact of chemical fixation on the microanatomy of mouse organotypic hippocampal slices. eNeuro, 10 (9), [ENEURO.0104-23.2023]. (doi:10.1523/ENEURO.0104-23.2023).

Record type: Article

Abstract

Chemical fixation using paraformaldehyde (PFA) is a standard step for preserving cells and tissues for subsequent microscopic analyses such as immunofluorescence or electron microscopy (EM). However, chemical fixation may introduce physical alterations in the spatial arrangement of cellular proteins, organelles, and membranes. With the increasing use of super-resolution microscopy to visualize cellular structures with nanometric precision, assessing potential artifacts, and knowing how to avoid them, takes on special urgency. We addressed this issue by taking advantage of live-cell super-resolution microscopy that makes it possible to directly observe the acute effects of PFA on organotypic hippocampal brain slices, allowing us to compare tissue integrity in a “before-and-after” experiment. We applied super-resolution shadow imaging (SUSHI) to assess the structure of the extracellular space (ECS) and regular super-resolution microscopy of fluorescently labeled neurons and astrocytes to quantify key neuroanatomical parameters. While the ECS volume fraction (VF) and microanatomic organization of astrocytes remained largely unaffected by the PFA treatment, we detected subtle changes in dendritic spine morphology and observed substantial damage to cell membranes. Our experiments show that PFA application via immersion does not cause a noticeable shrinkage of the ECS in hippocampal brain slices maintained in culture, unlike the situation in transcardially perfused animals in vivo where the ECS typically becomes nearly depleted. Our study outlines an experimental strategy to evaluate the quality and pitfalls of various fixation protocols for the molecular and morphologic preservation of cells and tissues.

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ENEURO.0104-23.2023.full - Accepted Manuscript
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Accepted/In Press date: 28 July 2023
e-pub ahead of print date: 14 September 2023
Published date: 14 September 2023
Additional Information: Funding Information: This work was supported by ANR (Agence Nationale de la Recherche) Grants ERA NET NEURON, ANR-17-NEU3-0005, and ANR-17-CE37-0011 (to U.V.N.), Fondation pour la Recherche Médicale, the Human Frontier Science Program Grant RGP0041/2019, the European Research Council Synergy Grant ENSEMBLE, #951294 (to U.V.N.), and a PhD fellowship from Bordeaux Neurocampus Graduate Program (A.I.). S.B. received funding from Horizon 2020 program under the Marie Sklodowska-Curie Grant 794492 as well as from the Fonds AXA pour la Recherche, AXA Banque Direction Banque Patrimoniale et Ses Donateurs. M.A. received funding from Japan Society for the Promotion of Science (JSPS). Publisher Copyright: © 2023 Idziak et al.
Keywords: PFA fixation artefacts, STED microscopy, dendrites, extracellular space, micro anatomy, mouse brain tissue

Identifiers

Local EPrints ID: 482092
URI: http://eprints.soton.ac.uk/id/eprint/482092
ISSN: 2373-2822
PURE UUID: 10623a83-53a9-4354-99ac-33a3a8a62307
ORCID for V V G Krishna Inavalli: ORCID iD orcid.org/0000-0002-7100-0214

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Date deposited: 19 Sep 2023 16:34
Last modified: 06 Jun 2024 02:09

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Contributors

Author: Agata Idziak
Author: V V G Krishna Inavalli ORCID iD
Author: Stephane Bancelin
Author: Misa Arizono
Author: U Valentin Nägerl

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