Ab182136

Sections of 4 µm thickness were cut from formalin-fixed paraffin-embedded samples of mouse tumors at T10 for Ki-67 staining, and at T10 and T20 for β2- and β3-AR staining. Immunostaining was performed according to standard procedures. Briefly, sections were deparaffinized in xylol and hydrated with grade ethanol concentrations until distilled water. Antigen retrieval was performed by immersing the slides in a thermostat bath containing 10 mM citrate buffer (pH 6.0) for 15 min at 98 °C followed by cooling for 20 min at room temperature. Endogenous peroxidase activity was blocked by treating the sections with hydrogen peroxide blocking reagent (Thermo Fisher, Rockford, IL, USA). After blocking with normal horse serum (UltraVision, Thermo Fisher), sections were incubated with the following primary antibodies: Ki-67 (1:100, rabbit polyclonal, PA5-19462, Thermo Fisher), β2-AR (ab182136, Abcam, Cambridge, UK), and β3-AR (ab94506, Abcam, Cambridge, UK). Bound antibodies were visualized using aminoethyl carbazol or 3,3′-diaminobenzidine as chromogens. Nuclei were counterstained with Mayer’s hematoxylin. Negative controls were performed by substituting the primary antibody with a non-immune serum. Quantification of the optical density (OD) in each specimen was obtained by using the Immunohistochemistry (IHC) Image Analysis Toolbox of ImageJ 1.48v software (NIH, USA).

Foot sections were first subjected to three 10−15-min rinses with 0.01 M PBS containing 0.3% Triton X-100 (A600198, BBI) (PBST). The samples were then blocked in the immunostaining blocking buffer (no. E674004, BBI) for 1 h. Subsequently, the samples were incubated with primary antibodies with in primary antibody dilution buffer (no. E674004, BBI) at 4 °C overnight. After three rinses with PBS, the samples were reacted with fluorescent dye-conjugated secondary antibodies in immunostaining secondary antibody dilution buffer (no. E674005, BBI) at RT for 1.5 h. The samples were then well rinsed three times and carefully mounted with DAPI Fluoromount-G (no. 0100-20, Southern Biotech). The primary antibodies used in this study included the following: rat anti-CD68 (NBP2-33337, Novus Biologicals; 1:400), rabbit anti-IL-6 (ab290735, Abcam; 1:50), rabbit anti-ADRB2 (ab182136, Abcam; 1:100), rabbit anti-Cleaved Caspase-3 (Cat. # 9661 S, CST; 1:400). Secondary antibodies conjugated to Alexa Fluor 488 (AF488), and AF594 included the following: AF488 donkey anti-rat (34406ES60, YEASEN) and AF594 donkey anti-rabbit (A21207, Invitrogen) and were diluted 1:400 unless otherwise specified.

The brain samples were lysed in RIPA lysis buffer with protease inhibitor cocktail (B14001, Bimake) by a tissue homogenizer, followed by ultrasonication and centrifugation. The bicinchoninic acid method was used to determine the concentrations of proteins that were mixed with 5 × Laemmli sample buffer and denatured for 5 min at 95 °C. A total of 30 µg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were incubated with blocking buffer (TBST buffer containing 5% skim milk powder) for 60 min at room temperature. Next, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies were as follows: anti-FTO (1:1000, ab92821, Abcam), anti-ADRB2 (1:1000, ab182136, Abcam), anti-SIRT1 (1:1000, 8469 S, Cell Signaling Technology), anti-c-MYC (9402 S, 1:1000, Cell Signaling Technology), and anti-GAPDH (1:10000, GTX100118, GeneTex), HRP-conjugated secondary anti-rabbit (1:5000, GTX213110-01, GeneTex), and HRP-conjugated secondary anti-mouse (1:5000, GTX213111-01, GeneTex). After incubation for 1 h with the corresponding secondary antibodies, the protein bands were detected by chemiluminescence using an ECL reagent.