Ab24509

Paraffin-embedded kidney sections were deparaffinized, hydrated, antigen retrieved, and endogenous peroxidase activity was quenched by 3% vol./vol. H₂O₂. Sections were then blocked with 10% vol./vol. normal donkey serum, followed by incubation with anti-PPARα (ab24509, Abcam), anti-CPT1α (ab128568, Abcam), anti-HIF-1α (NB100-123, Novus, New York, NY, USA), anti-HK2 (ab76959, Abcam), or anti-phospho-LDH (8176S, Cell Signaling Technology) overnight at 4 °C. After wash, sections were incubation with secondary antibody for 1 h, followed by incubation with avidin–biotin complex reagents for 1 h at room temperature before being subjected to substrate 3-amino-9-ethylcarbazole (Vector Laboratories, Burlingame, CA, USA). The percentage of positive cells to the selected field was analyzed using Image Pro Plus 6.0 software. An average percentage for each section was calculated. At least ten randomly chosen fields under the microscope were evaluated for each sample, and an average score was calculated.

The sample protein was extracted from liver tissues after the cells were lysed by RIPA buffer (89901, Thermo Scientific) at 4°C for 20 minutes and centrifuged for 10 minutes (4°C, 11180.0g). The secondary antibodies were anti‐PPARγ (ab209350, Abcam), anti‐PPARα (ab24509, Abcam), anti‐FXR (NBP2‐16550, Novus), anti‐RARα (ab254098, OriGene) and anti‐PPARδ (ab23673, Abcam). The protein was quantified completely by automatic Western blotting through a Wes automated system (ProteinSimple). The dynamic linear range of the standard protein was diluted in a gradient from 0.125 to 4 mg/mL. The Western blotting process was conducted according to the instructions for the Wes system, which differs from the traditional method. The machine was run with default parameters, and imaging and analysis were performed using Compass software (ProteinSimple).

Total protein samples were isolated from liver tissue and cells cultured using RIPA lysis buffer (Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (Roche, Basel, Switzerland). Samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Millipore, CA). The membranes were blocked in 5% milk and incubated with primary antibodies against mTOR (2983T), p-mTOR (5536T), p70S6 (9234), S6 (2217), pS6 (2211), TLR2 (13744), pNF-κBp65 (3033), NF-κBp65 (4764), NF-κBp50 (13586) and β-actin (4970) (CST, USA) at 1:1000 in TBST. Antibodies against PPAR-α (ab24509), SREBP-1 (ab28481), FASN (ab22759), ACC1 (ab45174), S6K1 (ab32529) and Caspase-1 (ab1872) were obtained from Abcam (Cambridge, USA). The intensity of the bands was analyzed by scanning densitometry and quantified by NIH Image J software. For protein expression, the specific band intensity was quantified, normalized to those of β-actin, and presented as a value relative to the control.

Protein abundance was determined in AT samples (n = 9 per group in S vs. W, and n = 6 per group in CL vs. HS) by immune blotting method. The protein concentrations of AT were measured by Bradford assay (Bio-Rad protein quantification kit). Subsequently, under reducing conditions, SDS-PAGE was used to resolve 20 g of sample in Laemmli loading buffer, which was then transferred to nitrocellulose membrane with the following antibodies: MGLL (1:200, ab24701, Abcam, Cambridge, UK), β-actin (1:1000, ab46805, Abcam Biotech, Cambridge, UK), CB1 (1:200, ab23703, Abcam Biotech, Cambridge, UK), FAAH (1 µg/mL, ARP33121_P050, Aviva Systems Biology, San Diego, CA, USA), CB2 (ADI-905-820-100, Enzo, Farmingdale, NY, USA), PPAR-α (ab24509, Abcam Biotech, Cambridge, UK), TRPV1 (1 µg/mL, WH0007442M1, Sigma, St. Louis, MO, USA) and TNF-α (OACA04183, Aviva Systems Biology, San Diego, CA, USA). For protein detection, a 1:10,000 dilution of goat anti-rabbit HRP conjugated secondary antibody (Jackson Immunoresearch 111-035-003, West Grove, PA, USA) was used for chemiluminescence reaction. ImageJ software was used to process and analyze the data (NIH, Bethesda, MD, USA). β-actin was used to equalize the signal bands.