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For the determination of the wet and dry weight, articular cartilage samples were separated from all the visible subchondral bone using a scalpel (n?=?5 per group), and weighed (wet weight). Subsequently, samples were snap frozen in liquid nitrogen, lyophilized (Martin-Christ, Germany), and weighed (dry weight). Lyophilized cartilage samples were digested with 0.5?mg/mL proteinase K (Invitrogen, Carlsbad, USA) at 60��C overnight. Absorbance was measured at 525?nm in a microplate reader (Biorad, Philadelphia, USA) after reaction with dimethylmethylene blue (Sigma�CAldrich), and GAG content was calculated using a standard of chondroitin sulfate C (Sigma�CAldrich)10. Statistical comparisons were made using a two-tailed Student��s t-test with significance determined by a P-value Obeticholic Acid smaller than 0.05. For comparisons of samples Doxorubicin scanned before and after fixation (Group III), paired t-tests were used. All data are represented as mean and 95% confidence interval (lower limit, upper limit). Analysis of variance (ANOVA) was used to compare Group I, Group II and Group III with significance determined by a P-value smaller than 0.05. The presence and zonal distribution of GAGs throughout the bovine articular cartilage tissue samples was confirmed by means of Safranin O staining [Fig.?2(A)], which is directly proportional to the GAG content9. The intensity of the staining in the cartilage tissue increased from the superficial layer towards the deeper layers. The average attenuation, as an indirect measure of overall GAG content of the unfixed samples, did not significantly differ between Group I and Group IIIa (n?=?8, 3667.3 (3357.6�C3977.0) HU vs 3618.1 (3248.0�C3988.1) HU, P?=?0.66). However, fixation FARP1 prior to EPIC-��CT imaging (Group II) resulted in a significant decrease of the attenuation by 14.3% compared to that of unfixed samples (Group I) (n?=?8, 3142.2 (2812.8�C3471.5) HU vs 3667.3 (3357.6�C3977.0) HU, P?