Transmission electron microscopy

Detecting the presence of autophagic vesicles by using transmission electron microscopy (TEM) is the most sensitive and gold standard technique to monitor autophagy. Control and analogue 2c treated (14.9 μM; 0–48 h) HT-29 cells (2.5 × 10⁵/ml) were fixed in 2.5 % glutaraldehyde and 2 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at 4 °C. After rinsing in PBS, cells were post fixed in osmium tetroxide (1 %) for 2 h, dehydrated in graded acetone and embedded in araldite CY212. Semi thin sections were cut, stained with 0.5 % toluidine blue (5 min) and examined under a light microscope (Olympus, 60 ×). Ultrathin sections were stained with 2 % uranyl acetate and Reynold’s lead citrate, and observed with a transmission electron microscope (Technai G2) [24].