Figure 1.

Identification and quantitation of autophagosomes in the liver of food-restricted mice. GFP-LC3 transgenic mice were food-restricted (food-rest) to activate autophagy, and 24 or 48 hours later, the liver was harvested and the induction of autophagy was analyzed in vibratome-cut sections by confocal microscopy. (A) representative flattened images of GFP-LC3 signal in hepatocytes are shown; sections were stained with phalloidin and Hoechst 33342 to label F-actin and nuclei, respectively. A merged fluorescent image for each mouse is shown in the right-hand column; GFP-LC3 (green), F-actin (red), nuclei (blue). Two sets of control mice were included: wild-type C57BL/6 mice were used to determine the background level of green fluorescence, which was very low (one advantage of vibratome sections); and normal-fed GFP-LC3 mice provided a baseline for autophagic activity in the liver. (B) Quantitative analysis of autophagosomes in hepatocytes, including the number of autophagosomes/cell, as well as their area, perimeter, feret and circularity [defined as 4π(area)/(perimeter)²]. Data are shown as the average + se of 80–134 cells from 3 or 4 mice per group; ^ap < 0.001, ^bp < 0.01, ^cp < 0.05.