Computational protocol: Blackcurrant Suppresses Metabolic Syndrome Induced by High-Fructose Diet in Rats

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Protocol publication

[…] Prior to immunohistochemical staining, tissue sections in paraffin were mounted on poly-l-lysine-coated slides (Fisher Scientific, Pittsburgh, PA, USA). The tissue on each slide was immunostained using Invitrogen HISOTO-STAIN-SP kits with the labeled streptavidin-biotin (LAB-SA) method. After antigen retrieval, slides were immersed in 3% hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity and rinsed with PBS. Next, slides were incubated with 10% nonimmune goat serum for 10 min at room temperature and incubated with primary antibodies against ICAM-1, VCAM-1, ET-1, and eNOS (1 : 200; Santa Cruz, CA, USA) in humidified chambers overnight at 4°C. Next, slides were incubated with biotinylated secondary antibodies for 20 min at room temperature, followed by incubation with horseradish peroxidase-conjugated streptavidin for 20 min at room temperature. Peroxidase activity was visualized using a 3,3′-diaminobenzidine (DAB; Novex, CA) substrate-chromogen system with hematoxylin counterstaining (Zymed, CA, USA). For quantitative analysis, the average score of 10–20 randomly selected areas was calculated using NIH Image Analysis Software, ImageJ (NIH, Bethesda, MD, USA). [...] All experiments were repeated at least 3 times. Results are expressed as mean ± S.D. or mean ± S.E.M. Data were analyzed using Sigmaplot 10.0 software. Student's t-test was used to determine significant differences between groups. Results of P < 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Hyperglycemia, Hypertension, Liver Diseases, Metabolic Diseases, Dyslipidemias
Chemicals Acetylcholine, Bilirubin, Cholesterol, Fructose, Glucose, Nitroprusside