All the animal experiments were performed in accordance with the guidelines established by the Animal Ethics Committee of Wonkwang University, and this experiment was approved by the committee (Approval No. WKU15-101). To evaluate the effect of BGE on fasting blood glucose levels, C57BL/6J (dbdb) 30 mice (20 –25g, 4 weeks, male Lepr(+/+)) and C57BL/6J (dbh) 10 mice (20–25g, 4 weeks, male Lepr(+/-)) were used as the animal model. All the animals were purchased from SAMTACO (Osan, Korea) All the experiments in this study were conducted for 4 weeks. The mice were given free access to water and kept at a constant room temperature under a 12/12–hour light/dark cycle. The mice were adapted to new environment and food for 1 week before starting the
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The fixed tissues were processed routinely for paraffin embedding. Insulin and glucagon were detected in the 5-μM pancreatic sections by guinea pig anti-human insulin diluted by 1/100 (Millipore) and rabbit anti-glucagon diluted by 1/3000 (Millipore Corp., Billerica, MA, USA), respectively, followed by incubation with peroxidase-conjugated AffiniPure goat anti-rabbit Ig (H + L) diluted by 1/100 (Proteintech Group Inc., Chicago, USA). 3,3-DAB was applied to the sections as a substrate for peroxidase. The sections were counterstained with hematoxylin. To observe collagen content, picrosirius red staining was performed by using a picrosirius red staining kit (Polysciences, Inc. Warrington). The sections of muscle were deparaffinized and rinsed by distilled water. The washed sections were soaked in picrosirius red solution A for 2 min. The sections were rinsed again with distilled water, soaked in picrosirius red solution B for 60 min and picrosirius red solution C for 2 min, immersed in 70% alcohol for 45 s, dehydrated, cleared, and mounted. Image acquisition was performed using an optical microscope (Nikon Eclipse 80i; Tokyo, Japan) with a magnifying power of 400. For the size determination of islets of Langerhans, the surface area of islets of Langerhans was traced manually and analyzed using Image J software (n = 10 per group). The intensities of the immunostained signals for insulin and glucagon proteins were measured as
The fixed tissues were processed routinely for paraffin embedding. Insulin and glucagon were detected in the 5-μM pancreatic sections by guinea pig anti-human insulin diluted by 1/100 (Millipore) and rabbit anti-glucagon diluted by 1/3000 (Millipore Corp., Billerica, MA, USA), respectively, followed by incubation with peroxidase-conjugated AffiniPure goat anti-rabbit Ig (H + L) diluted by 1/100 (Proteintech Group Inc., Chicago, USA). 3,3-DAB was applied to the sections as a substrate for peroxidase. The sections were counterstained with hematoxylin. To observe collagen content, picrosirius red staining was performed by using a picrosirius red staining kit (Polysciences, Inc. Warrington). The sections of muscle were deparaffinized and rinsed by distilled water. The washed sections were soaked in picrosirius red solution A for 2 min. The sections were rinsed again with distilled water, soaked in picrosirius red solution B for 60 min and picrosirius red solution C for 2 min, immersed in 70% alcohol for 45 s, dehydrated, cleared, and mounted. Image acquisition was performed using an optical microscope (Nikon Eclipse 80i; Tokyo, Japan) with a magnifying power of 400. For the size determination of islets of Langerhans, the surface area of islets of Langerhans was traced manually and analyzed using Image J software (n = 10 per group). The intensities of the immunostained signals for insulin and glucagon proteins were measured as