RT Journal Article
SR Electronic
T1 The Impact of Chemical Fixation on the Microanatomy of Mouse Organotypic Hippocampal Slices
JF eneuro
JO eNeuro
FD Society for Neuroscience
SP ENEURO.0104-23.2023
DO 10.1523/ENEURO.0104-23.2023
VO 10
IS 9
A1 Idziak, Agata
A1 Inavalli, V. V. G. Krishna
A1 Bancelin, Stéphane
A1 Arizono, Misa
A1 Nägerl, U. Valentin
YR 2023
UL http://www.eneuro.org/content/10/9/ENEURO.0104-23.2023.abstract
AB 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.