Autophagy plays a key role in maintaining cellular homeostasis and is frequently altered
in cancer cells. Extracts of the human amniotic membrane (hAM) exhibit antiinflammatory
and antitumour effects, yet their impact on autophagy in urothelial cells
remains unclear. The aim of this thesis was to evaluate the suitability of correlative
microscopy (CLEM), which combines light and electron microscopy of the same cellular
region, for identifying autophagosomal compartments in T24 cancerous urothelial cells
after 24 h of treatment with an hAM extract. Cells were chemically fixed, embedded in
gelatin and processed by the Tokuyasu method into cryo-ultrathin sections, on which the
autophagy marker LC3B (Alexa Fluor 555) was immunolabelled and nuclei were stained
with Hoechst. The same regions were imaged sequentially by confocal and transmission
electron microscopy and the micrographs correlated using ImageJ/Fiji software and the
BigWarp plugin. Across seven immunolabelling runs (18 grids and 7 correlated samples
in total), Hoechst reliably labelled nuclei and enabled correlation between the light and
electron planes, whereas the LC3B signal was weak and largely non-specific. The
electron-microscopy was mostly unsuccessful unambiguous identification of doublemembrane
vesicles. Because of these limitations and the artefacts arising during
Tokuyasu sample preparation (non-specific antibody binding, section folding and tearing,
and flattened cell morphology), formal quantification of autophagosomes was not
performed. The results indicate that, in its current form, the method is not yet optimised
for quantitative analysis of autophagosomal compartments in T24 cells, but they provide
a methodological basis and guidance for further protocol optimisation and its combination
with molecular methods (western blot, qPCR).
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