The Impact of Chemical Fixation on the Microanatomy of Mouse Organotypic Hippocampal Slices

Agata Idziak; V. V. G. Krishna Inavalli; Stéphane Bancelin; Misa Arizono; U. Valentin Nägerl

doi:10.1523/ENEURO.0104-23.2023

Article Figures & Data

Figures

Tables

Download figure
Open in new tab
Download powerpoint
Figure 1.
Brief PFA incubation does not affect ECS volume fraction. A, Graphical overview of the workflow of experiments and analysis. B, Time-lapse shadow imaging of ECS in live and buffered PFA conditions. The live condition is represented both with a raw and inverted LUT. C, ECS volume fraction α does not change during a 30-min incubation with PFA. The images were analyzed either as a whole (“global”) or divided into “neuropil” or “cell bodies” areas (n = 6; presented as mean + SD). D, Representative images of ECS live and after 30 min of incubation with PFA. Blue squares indicate the magnified area that is shown below with an inverted LUT. E, Paired analysis of ECS volume fraction live and after 30 min of incubation with PFA (n = 5; ns: not significant; p > 0.05; in paired t-test). Scale bars: 10 μm.
Download figure
Open in new tab
Download powerpoint
Figure 2.
Prolonged PFA incubation introduces artifacts into ECS structure. A, Representative images of ECS live and after 90 min of incubation with PFA. The inset and white arrow indicate “cell blebbing.” B, Left, Paired analysis of ECS volume fraction between live and 90 min after PFA fixation. The images were analyzed either as a whole (“global”) or divided into “neuropil” or “cell bodies” areas (n_cntrl = 6; n_PFA = 6; ns: not significant; p > 0.05; in Wilcoxon matched-pairs test). Right, Comparison of the “cell blebbing” between live and 90-min PFA conditions (n = 6; *p < 0.05; Mann–Whitney test). C, Representative STED images of ECS live and after 90 min of PFA fixation. D, SUSHI-based analysis of ECS widths. The blue lines indicate an example of the analyzed width. The line profiles are shown together with measured FWHMs. E, Paired analysis of ECS widths live and 90 min after PFA fixation (n_cntrl = 12; n_PFA = 16; ns: not significant; p > 0.05; paired t test). F, Representative images of ECS live and 180 min after PFA fixation. Inset and white arrows indicate examples of dye accumulation around cell bodies. G, A representative confocal shadow image after fixation overnight with PFA. Scale bars (A, F, G): 10 μm; (C, D): 5 μm.
Download figure
Open in new tab
Download powerpoint
Figure 3.
PFA fixation has no detectable effects on astrocytic fine structure. A, Representative confocal images of a brain slice expressing GFAP-Clover in astrocytes, live and 90 min after PFA fixation. White arrows indicate a representative astrocytic main branch and cell body. B, Paired analysis of astrocytic areas of main branches and cell bodies live and 90 min after PFA fixation (n_branches = 12; n_bodies = 11; ns: not significant; p > 0.05; paired t test). C, Representative STED images of astrocytic structures in spongiform domain expressing GFAP-Clover, live and 90 min after PFA fixation. The blue lines show representative line profiles of astrocytic structures to determine their width. The line profiles are shown on the right together with calculated FWHMs. D, Paired analysis of widths of astrocytic fine processes, live and 90 min after PFA fixation (n_cntrl = 26; n_PFA = 28; ns: not significant; p > 0.05; paired t test). Scale bars: 10 μm.
Download figure
Open in new tab
Download powerpoint
Figure 4.
PFA fixation affects spine morphology. A, Representative STED maximum intensity z-projections of a dendritic segment cytosolically filled with Citrine, live and 90 min after PFA fixation. White arrow indicates a “dendritic vacuole.” B, Bar graph showing appearance of “dendritic vacuoles” after 90 min under live or fixed conditions (expressed as appearances over total number of experiments: control: 2 out of 23; PFA: 15 out of 31; p = 0.0024; bars show mean + SD; Mann–Whitney test). C, Representative STED images of dendritic spines and an example of head and neck analysis using SpineJ. D, Paired analysis of spine head area and neck length (n_cntrl = 79; n_PFA = 86; ns: not significant; p > 0.05; *p = 0.0157; Wilcoxon matched-pairs test). E, An example of a dendritic spine with a wider neck after 90 min of PFA fixation. F, Paired analysis of spine neck width (smallest and median values) between live and 90-min PFA-fixed conditions (n_cntrl = 79; n_PFA = 86; ns: not significant; ****p < 0.0001; Wilcoxon matched-pairs test). Scale bars (A): 10 μm; (C, E): 500 nm.

Tables

Figures

View popup

Table 1

PFA fixation-induced effects on brain tissue microstructure

Time of fixation Structure	30 min	90 min	>90 min
Extracellular space	No change in VF	No change in VF No change in width	NE (dye uptake)
Global tissue	No visible changes	Cell blebbing	Membrane permeabilization
Astrocytes	NE	No changes in areas of cell bodies and main branches No changes in widths of fine processes	NE
Dendrites Dendritic spines	NE	“Dendritic vacuoles” No changes in spine neck length Decreased spine head area Increased spine neck width	NE

Summary of observed changes in various tissue structures (ECS, astrocytes and dendrites) for different fixation times. VF: volume fraction; NE: not examined.