Cell signalling
Does chemical fixation squash the living cortex?
Open access · cc by · source: Europe PMC
In adult mouse barrel cortex, aldehyde perfusion shrank tissue ~30% and left only 2.5% extracellular space versus 15.4% after high-pressure freezing; cryo tissue also had smaller astrocyte volume, looser glial coverage, and more docked vesicles.
Study at a glance
- Design
- Animal / in-vitro — Quantitative EM comparison of adult mouse somatosensory neocortex after aldehyde perfusion versus high-pressure freezing/freeze substitution
- N
- N=4 · Primary neuropil comparisons N=4 mice per fixation; cortical thickness used N=6 fresh vs 14 chemically fixed; vessel coverage n=11 cryo vs 69 perfused
- Population
- Adult C57BL/6 mice (7–10 weeks), primary somatosensory barrel cortex
- Outcome
- Tissue shrinkage, extracellular-space and astrocyte volume fractions, synapse density/coverage, vesicle shape and docking
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Key findings
Cortical thickness −16%; ~30% volume loss. ECS 15.4 ± 5.4% cryo vs 2.47 ± 1.5% chemical (p=0.003). Astrocyte volume 7.4% vs 14.4% (p=0.01). Synapse density 0.63 vs 0.87 µm⁻³ (p=0.042). Perisynaptic gap 166 vs 53 nm. Capillary astrocyte coverage 63% vs 94%. Cryo revealed more docked vesicles; flattened ‘type 2’ vesicles were a chemical-fixation artifact.
Methodology
Compared fresh vs chemically fixed cortical thickness, then serial-section EM of high-pressure-frozen vs aldehyde-perfused neuropil (N=4 mice/group), scoring ECS, astrocyte/neurite volumes, synapse density, perisynaptic gaps, astrocytic coverage of synapses and capillaries, and vesicle geometry.
Limitations
High-pressure freezing still samples tiny freshly cut pieces, so it is not a whole-brain connectomics method; ‘more native’ does not prove every cryo number matches living cortex under all conditions.
How this study connects
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