Sc34285

The cell homogenates were loaded into 10% SDS-polyacrylamide gels and subjected to electrophoresis. The separated proteins were transferred to nitrocellulose membranes and then incubated with bovine albumine serum solution for 1 hour, followed by overnight incubation at 4°C with the following antibodies: mouse monoclonal β-actin antibody (diluted 1:5000; AC5441 –Sigma Aldrich, St. Louis, MO, USA), goat polyclonal CAT antibody (CAT; diluted 1:1000; sc34285—Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit polyclonal PGC-1α (dilution 1:2500; ab191838—Abcam, Cambridge, UK); rabbit polyclonal SOD2 (diluted 1:2000; ab13534—Abcam, Cambridge, UK), or with rabbit polyclonal SIRT3 (diluted 1:1500; ab189860—Abcam, Cambridge, UK). Protein detection was carried out using secondary infrared fluorescent dye conjugated antibodies absorbing at 800 nm or 700 nm. The blots were visualized using an Odyssey Infrared Imaging Scanner (Li-Cor Inc., Lincoln, USA).

Immunohistochemistry (IHC) was performed using the EnVision FLEX Mini Kit (K8024, Agilent-Dako, Santa Clara, CA, USA) and Dako Autostainer system. Paraffin-embedded sections of epigastric arteries (3 µm thick) were deparaffinised and rehydrated. Heat epitope retrieval was performed at 95 °C for 20 min with a pH 9 (K8004, Agilent-Dako, Santa Clara, CA, USA).
Endogenous peroxidase activity was blocked using EnVision™ FLEX Peroxidase-Blocking Reagent (DM821). The sections were then incubated with polyclonal goat anticatalase antibody (SC34285, 1/100; Santa Cruz Biotechnology, Dallas, TX, USA) or polyclonal rabbit anti-RUNX2 antibody (SC10758, 1/50; Santa Cruz Biotechnology, Dallas, TX, USA) for 30 min, diluted in EnVision™ FLEX Antibody Diluent (K8006, Agilent-Dako, Santa Clara, CA, USA).
For catalase IHC, a polyclonal rabbit anti-goat (P0449) secondary antibody was used, while for RUNX2, the Secondary Dako EnVision + Dual Link System-HRP (Agilent-Dako, Santa Clara, CA, USA) was used, both for 30 min at room temperature. The signal was detected using diaminobenzidine chromogen as substrate in Dako EnVision™ FLEX/HRP (DM822, Agilent-Dako, Santa Clara, CA, USA). Sections were counterstained with haematoxylin. Negative controls were processed by omitting the primary or secondary antibody.