Dodecenyl succinic anhydride

ANT2-OE or mCherry-OE senescent HDFs and proliferating HDF (young cell) pellets were obtained and fixed in 2.5% glutaraldehyde (in 0.1 M sodium cacodylate buffer) for 1 hour at room temperature. After washing with cacodylate buffer, the pellets were embedded in low-melting-point agarose (number 4250-050-02, Trevigen, Gaithersburg, MD) and cut into small pieces. Skin tissue samples from old mice infected with lentivirus for overexpressing mCherry or ANT2 and those from young mice were fixed in 2.5% glutaraldehyde and washed with 0.1 M cacodylate buffer. The fixed samples were incubated in 1% osmium tetroxide overnight at 4 C. After washing with cacodylate buffer, the samples were dehydrated with a series of graded ethanol and 100% propylene oxide. The samples were then embedded in medium-intensity resin (nadic methyl anhydride, EPON epoxy, dodecenyl succinic anhydride, and benzyldimethylamine, Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were prepared using a cryo-ultramicrotome (EM UC7, Leica, Wetzlar, Germany) and stained with lead citrate and uranyl acetate. The sections were then imaged with a Bio-transmission electron microscope (FEI Tecnai G2 F20 TWIN TMP, Amsterdam, Netherlands).