The concentrations of serum biomarkers were determined using enzyme-linked immunosorbent assay kits (adiponectin, Boster, Pleasanton, CA, United States; leptin, Boster, Pleasanton, CA, United States; high-sensitivity C-reactive protein (HS-CRP), Roche, Basel, Switzerland; TNF-α, Immunite 1000 LKNF1, Siemens Medical Solutions Diagnostics, Llanberis, UK) [24 (link),25 (link)]. The serum level of 25(OH)D was measured using an electro-chemiluminescence immunoassay (Cobas® Vitamin D3 assay, Roche Diagnostics GmbH, Mannheim, Germany) with an interassay coefficient of variation of 2.2–13.6% [19 (link)]. Each biomarker assay was performed in duplicate according to the manufacturer’s instructions, and the mean value was used for further statistical analysis.
Adiponectin
Manufactured by Boster Bio
Sourced in United States
Adiponectin is a protein hormone that regulates glucose and lipid metabolism. It is secreted by adipose tissue and plays a role in the modulation of insulin sensitivity and energy homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Hscrp, by Roche (1 mentions)
Cobas vitamin d3 assay, by Roche (1 mentions)
Immunite 1000 lknf1, by Siemens (1 mentions)
Myostatin, by R&D Systems (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Tgf β, by R&D Systems (1 mentions)
Diaminobenzidine tetrahydrochloride, by Merck Group (1 mentions)
Primo star, by Zeiss (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
4 protocols using adiponectin
1
Serum Biomarker Analysis via ELISA
2
Serum Biomarker Analysis Protocol
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The concentration of circulating biomarkers was analyzed using serum samples in duplicate for each time point. Each biomarker analysis was conducted according to the manufacturer’s instructions. An enzyme-linked immunosorbent assay (ELISA) was performed to measure the circulating levels of myostatin (R&D Systems, Minneapolis, MN, USA), TGF-β (R&D Systems), TNF-α (Cohesion Bioscience, London, UK), IGF-1 (R&D Systems), adiponectin (Phoenix, NY, USA), and decorin (Boster Bio, Pleasanton, CA, USA).
3
Metabolic and Inflammatory Markers
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At baseline and after intervention, fasting blood samples were collected and serum was isolated and preserved at −70°C until the last sample was collected. Biochemical parameters such as fasting blood sugar (FBS), uric acid, lipid profiles (total cholesterol, triglycerides, low-density lipoprotein-cholesterol [LDL-C], high-density lipoprotein-cholesterol [HDL-C]) and hemoglobin A1C (Hb A1C) were measured using an auto-analyzer (Hitachi 911, Japan). Three inflammatory markers such as hs-CRP, IL-6, and TNF-α (AviBion, Vantaa, Finland) serum insulin and adiponectin (Boster Biological Technology, Pleasanton CA, USA) were measured by the enzyme-linked immunosorbent assay method.
4
Histological Analysis of Atherosclerotic Plaques
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Frozen plaques were sectioned crosswise over their longitudinal axis using a cryostat, and the major segment was used for histological analysis.
Alizarin Red S (Sigma, Missouri, USA) histological staining was used for calcium determination. The protocol was adapted (no de-paraffinization needed, slides were slowly immersed in distilled water) from IHC World website (19 ).
Immunohistochemical staining of the plaques to identify a proinflammatory/unstable profile was also performed with CD3 (eBioscience, San Diego, USA), CD68 (eBioscience, San Diego, USA) and Adiponectin (Boster Biological Technology, Pleasanton, USA) antibodies. Tissue sections were incubated with the primary antibody and with EnVision+ (Dako, Glostrup, Denmark). Color was developed in solution containing diaminobenzidine-tetrahydrochloride (Sigma, Missouri, USA), 0.5% H2O2 in phosphate-buffered saline buffer (pH 7.6). Slides were counterstained with hematoxylin and mounted (20 (link)).
Histological and immunohistochemical evaluations were performed using a semi-quantitative score of 0 to 3 (0–0 to 10% staining; 1–10 to 50% staining; 2–50 to 75% staining; 3–more than 75% staining). Slides were observed in a ZEISS Primo Star (ZEISS, Oberkochen, Germany) microscope.
Alizarin Red S (Sigma, Missouri, USA) histological staining was used for calcium determination. The protocol was adapted (no de-paraffinization needed, slides were slowly immersed in distilled water) from IHC World website (19 ).
Immunohistochemical staining of the plaques to identify a proinflammatory/unstable profile was also performed with CD3 (eBioscience, San Diego, USA), CD68 (eBioscience, San Diego, USA) and Adiponectin (Boster Biological Technology, Pleasanton, USA) antibodies. Tissue sections were incubated with the primary antibody and with EnVision+ (Dako, Glostrup, Denmark). Color was developed in solution containing diaminobenzidine-tetrahydrochloride (Sigma, Missouri, USA), 0.5% H2O2 in phosphate-buffered saline buffer (pH 7.6). Slides were counterstained with hematoxylin and mounted (20 (link)).
Histological and immunohistochemical evaluations were performed using a semi-quantitative score of 0 to 3 (0–0 to 10% staining; 1–10 to 50% staining; 2–50 to 75% staining; 3–more than 75% staining). Slides were observed in a ZEISS Primo Star (ZEISS, Oberkochen, Germany) microscope.
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