Ab45422

Rb1 (>98%, ab142646) was purchased from Abcam (Cambridge, UK). 3-Isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), insulin, and Oil Red O powder were purchased from Sigma (St. Louis, MO, United States). L748337 was from Tocris Bioscience (Bristol, UK). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from Corning (NY, United States). Antibodies for liver kinase B1 (LKB1) (3047S), pLKB1 (Ser428) (3482S), AMP-activated protein kinase alpha (AMPKα) (2532S), pAMPKα (Thr172) (2535S), acetyl-CoA carboxylase (ACC) (3676S), pACC (Ser79) (3661S), silent information regulator T1 (SIRT1) (8469S), SIRT3 (5490S), phospho-hormone sensitive lipase (pHSL) (Ser563) (4139S), phospho-PKA substrate (9624S), UCP1 (14670S), and β-actin (3700S) were purchased from Cell Signaling Technology (Beverly, MA, United States); the antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sc-32233) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States); antibodies for PGC1α (ab54481), comparative gene identification 58 (CGI58) (ab59488), adipose triglyceride lipase (ATGL) (ab207799), HSL (ab45422), and β3AR (ab94506) were purchased from Abcam (Cambridge, UK); the antibody for PPARα (PA1-822A) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).

10 µm serial cuts were performed on the same tissue samples embedded in paraffin used for H&E staining. UCP1, PGC1 alpha and HSL expression were studied by means of immunohistochemistry. Briefly, hRAN and hRAT microtome slides were first deparaffinized, and then a heat-mediated antigen retrieval, endogenous peroxidase blocking and nonspecific tissue blocking were performed. Slides were then incubated with the different primary antibodies (Anti-UCP-1. SIGMA U6382. Dilution of 1:500; Anti-PGC1 alpha. Abcam ab54481. Dilution of 1:300; and Anti-HSL. Abcam ab45422. Dilution of 1:300) at 4 °C. And after that with an anti-rabbit biotinylated secondary IgG antibody. Finally, slides were incubated with peroxidase-conjugated streptavidin. Peroxidase reaction was performed with chromogen 3,3′-diaminobenzidine (DAB) (DAKO LSAB + Kit, HRP). Hematoxylin counterstaining was performed. Serial cuts incubated in the absence of the primary antibody were used as negative controls. Images were taken with a Nikon Eclipse E200 Microscope fitted with a Micrometric SE Premium (Nikon Corp., Japan) digital still camera at 10× and 40× magnification. DAB staining quantification in the three tissue types was performed in 5 fields of each preparation as mentioned above^{3 (link)–5 (link)}.

Soleus muscle from each group was homogenized in a 1:10 (w:v) ratio of homogenizing buffer (100 mmol/L NaCl, 50 mmol/L Tris base, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA and 1% Tritonx100, pH 7.5), and a Bradford assay was used to determine total protein content. Polyacrylamide gels were composed of 10% acrylamide separating gel and 4% acrylamide stacking gel. Membranes were blocked in 5% non-fat dry milk and Tris buffered saline (TBS), then incubated overnight at 4 °C in anti-hormone sensitive lipase (HSL) antibody (1:1000) (ab45422, Abcam, Cambridge MA, USA). Following secondary antibody incubation (BioRad goat-anti-rabbit IgG (H + L)- HRP conjugated 1662408, and goat-anti-mouse IgG (H + L)-HRP conjugated 1721101 as per manufactures instructions), membranes were washed and visualized using a luminol-based chemiluminescent substrate (BioRad Western C Enhanced Chemiluminescent Kit, 170-5070) on a BioRad Chemidoc XRS imager. Densities were determined using Quantity One software.