Figure 7. Autophagosomes accumulate during neuronal senescence. (A) Top row, autophagosomes were stained with CytoID® and nuclei with Hoechst in primary culture of cortical cells incubated during the indicated DIV; scale bars represent 15 μm. Middle rows, immunofluorescence to detect LC3 in neurons (expressing βIII-TUBULIN) of primary cortical cells cultivated during the indicated DIV. Scale bars represent 5 μm. Bottom rows, electron micrographs showing accumulation of autophagosomes in 26 DIV cortical cells, detected by immunogold localization of LC3 (arrow heads). Squares indicate the amplified area below. (B) Immunofluorescence to detect p62/SQSTM1 in cortical cells cultured during the indicated DIV. Nuclei were stained with DAPI. Notice that p62/SQSTM1 in neurons (expressing βIII-TUBULIN) accumulated at 26 DIV. Scale bars represent 5 μm. (C) Immunofluorescence to simultaneously detect LC3 and Lamin-A/C to observe intranuclear folds as a senescence marker, in cortical cells cultured during the indicated DIV. Scale bars represent 25 μm. (D) LC3 and p62/SQSTM1 also accumulate in cortical neurons (expressing βIII-TUBULIN or MAP2) form old rat brains. Scale bars represent 30 μm.