Sightx viewer software

OSCs were fixed with ITO-fixing solution (25% paraformaldehyde, 25% Glutardialdehyde, 0.1 M cacodylatbuffer) and pre-embedded horizontally in HistoGel. The OSCs were then processed for Epon embedding following standard protocols. Ultrathin cross sections were cut with a microtome (Ultracut Leica, Jena, Germany) and analyzed with a JEM-1400Plus (JEOL, Tokyo, Japan). SightX-Viewer software (Jeol, version 1.2.3.537) was used to evaluate the morphological changes.

The diameter of individual autophagic compartments (autophagosomes and autolysosomes) was determined in mRFP-EGFP-LC3-expressing cells, imaged by fluorescence microscopy (SIM and confocal microscopy). Specifically, the full width at half maximum of the red fluorescence (mRFP) signal intensity was measured using ZEN microscope software (Zeiss, Oberkochen, Germany). All vesicle diameter values represent an average of two independent measurements made in the equatorial plane in two perpendicular directions (horizontally and vertically) to improve the accuracy of the measurement. We measured the diameter of all autophagic compartments that were in focus in the equatorial z stack of individual imaged cells.
The size of the autophagic compartments was also determined on TEM micrographs. Autophagy-related structures were identified based on specific morphological characteristics, such as spherical structures with a double-layered membrane containing different intercellular material, and their diameter was measured using SightX Viewer software (JEOL, Tokyo, Japan).