Ab 108327

After the last PET scan, mice were sacrificed, extremities fixed in 4 % PBS buffered formalin for 48 h, decalcified in Osteosoft (Merck, Darmstadt, Germany) for 4 weeks according to the manufacturer’s instructions, and embedded in paraffin. Sagittal sections of 4-μm thickness were cut, and consecutive sections stained with Movat pentachrome staining was performed using a staining kit (Morphisto, Frankfurt am Main, Germany). For immunohistochemistry (IHC), serial sections were stained using a Bond RXm system (Leica, Wetzlar, Germany, all reagents from Leica, unless otherwise indicated) with primary antibodies against α5-integrin (abcam, ab 108327, diluted 1:10.000) or β3-integrin (Cell Signaling, 13166S, diluted 1:300). Briefly, slides were deparaffinized using deparaffinization solution, pretreated with Epitope retrieval solution 2 (corresponding to EDTA buffer pH 8) for 30 min. Antibody binding was detected with a polymer refine detection kit without post-primary reagent and visualized with DAB as a dark brown precipitate. Slides were scanned (Leica AT2, Leica, Wetzlar, Germany) and evaluated using Imagescope Software (Leica, Wetzlar, Germany).

Tissues were snap-frozen in liquid nitrogen, ground using Cryomill in liquid nitrogen at 25 Hz for 2 min, and then lysed in Tris lysis buffer (1M Tris-HCl, 1M NaCl, 0.1M EDTA, 10% NP40). Protein concentrations were measured using the Bio-Rad BCA reagent. Samples were probed with antibodies against Atg7 (Sigma Aldrich, A2856, RRID:AB_1078239), Lkb1 (Santa Cruz Biotechnology, sc-32245, RRID:AB_627890), LC3 (Novus Biologicals, NB600-1384, RRID:AB_669581), p62 (American Research Products, 03-GP62-C, RRID:AB_1542690), p-ACC^S79 (Cell Signaling, 3661, RRID:AB_330337), ACC (Cell Signaling, 3676, RRID:AB_2219397), Atg5 (Abcam, ab108327, RRID:AB_2650499), p-S6^S235/236 (Cell Signaling, 4858, RRID:AB_916156), S6 (Cell Signaling, 2217, RRID:AB_331355), and β-actin (Sigma Aldrich, A1978, RRID:AB_476692). Western blots were quantified with Image J (National Institutes of Health). The intensities of bands were used to calculate relative ratios of the indicated protein over loading control (β-actin), which were then normalized based on the corresponding ratio in the wild type control sample.