Em420 transmission electron microscope

Cells were harvested, pelleted and fixed in paraformaldehyde (0.1% glutaraldehyde in 0.1 M sodium cacodylate) for 2 h, postfixed with 1% OsO₄ for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95–100% alcohol) and embedded in Epon-Araldite resin (Canemco, 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope. Cells with autophagic vacuoles were defined as positive if they had 5 or more autophagic vacuoles. The area occupied by autophagic vacuoles and the cytoplasm were determined with Image Pro Plus Image Analysis Software version 3 and used to calculate the cytoplasmic area occupied by the autophagic vacuoles [50] (link).

TEM was performed to observe autophagic vacuole ultrastructure as described before.^[42] NSCLC cells or tissue samples (≈1 mm³) were fixed in 2.5% glutaraldehyde at 4 °C overnight and washed with PBS twice and fixed in osmic acid for 2 h. Then samples were dehydrated by gradients of ethyl alcohol and acetone, embedded and saturated, sliced into ultrathin sections. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with an EM420 transmission electron microscope (Philips, Eindhoven, The Netherlands).