Ultrastructural analysis of adult mouse neocortex comparing aldehyde perfusion with cryo fixation
- Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
Figures
Figure 1 with 2 supplements
Chemical fixation reduces cortical volume and extracellular space.
(A) Coronal sections of fresh (left) and chemically fixed (right) adult mouse brains. Double-headed arrows overlaying the somatosensory cortex of each section show the position at which the cortical …
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Figure 1—source data 1
Data values and statistics underlying Figure 1B, D, E.
- https://doi.org/10.7554/eLife.05793.004
Figure 1—figure supplement 1
Schematic scale model (upper image) representing the fresh somatosensory cortex (outer cube) and chemically fixed cortex (inner cube), showing the extent to which the two fixations change the volume of this brain region.
Graph (lower left) showing the percentage of neuropil volume occupied by astrocytic (marked ‘Glia’) elements and ECS, in the cryo and chemically fixed neuropil. Graph (lower right) shows the …
Figure 1—figure supplement 2
Comparison between chemical fixation (left hand images; A and C) and cryo fixation (right hand images; B and D) of acute brain slices shows that both fixation conditions are able to reveal significant amounts of extracellular space.
However, this can only be clearly seen within 10 microns of the slice surface (B). Deeper into the cryo-fixed slice, the cellular elements appear disrupted with a fine latticed patterning indicating …
Figure 2
Cryo fixation reveals a larger peri-synaptic space and reduced astrocytic coverage.
(A) Cryo-fixed neuropil shows synaptic contacts with large amounts of surrounding extracellular space. (B) Synaptic density measurements show the chemically fixed neuropil to have 38% more synapses …
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Figure 2—source data 1
Data values and statistics underlying Figure 2B.
- https://doi.org/10.7554/eLife.05793.008
Figure 3
Cryo-fixed capillaries show less astrocytic coverage.
(A) Electron micrographs of transversely sectioned capillaries show the astrocytic endfeet pseudo-coloured in orange. Cryo-fixed example shows a darkly stained erythrocyte within the vessel lumen. (B…
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Figure 3—source data 1
Data values and statistics underlying Figure 3C.
- https://doi.org/10.7554/eLife.05793.010
Figure 4 with 1 supplement
Vesicles of symmetric synapses are distorted by chemical fixation.
(A) Cryo-fixed synapses on a dendritic shaft (left image) and on a dendritic spine (middle image) show similar rounded vesicles. A chemically fixed, high-pressure frozen and cryo-substituted (right …
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Figure 4—source data 1
Statistics underlying Figure 4B.
- https://doi.org/10.7554/eLife.05793.012
Figure 4—figure supplement 1
Examples of glutamatergic synapses (A, B, C), situated on dendritic spines, with round clear vesicles; and presumed GABAergic synapses (D, E, F) on dendritic shafts showing flattened, dark vesicles.
Scale bar is 200 microns.
Figure 5 with 1 supplement
Cryo fixation preserves larger numbers of vesicles at the pre-synaptic membrane.
(A) Electron tomography of a 200-nm thick section shows a cryo-fixed (upper) synapse with a large number of vesicles close to the presynaptic membrane in comparison with a similar chemically fixed …
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Figure 5—source data 1
Data values and statistics underlying Figure 5B.
- https://doi.org/10.7554/eLife.05793.015
Figure 5—figure supplement 1
Synapses found on dendritic spines were the same length.
Data are represented as mean ± SD. Unpaired Student's t-test assessed statistical significance; p = 0.561.
Author response image 1
Electron micrograph showing the neuropil from an acute slice chemically fixed immediately after it was vibratome sectioned. The image is taken 50 micons from the slice surface.
