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The Picrosirius Red imparts a distinct red colour to collagen fibres, facilitating morphometric analysis of the collagenous matrix area fraction from transverse sections of the LV using ImageJ software (National Institutes of Health, Bethesda, MD, USA; http://www.nih; Fujii et al. 2002). Confocal microscopy was used to determine the labelling pattern and signal intensity of ��-skeletal muscle actin and also for calculation of myocyte size from fluorescein isothiocyanate-conjugated lectin (L4895; Sigma Aldrich)-labelled paraffin-embedded sections (see Supplementary Methods). (-)-p-Bromotetramisole Oxalate For Western blotting, ventricle protein homogenates were prepared from frozen sections as previously described (Zheng et al. 2005), and 60��g of extract was resolved by 4�C15% SDS-PAGE. ��-Actin was used as a loading control, and membranes were incubated with antibodies as given in Table S1. Flash-frozen LV samples were also homogenized for nucleic acid extraction using an RNeasy kit (Qiagen, Valencia, CA, USA), and cDNA was generated with a Superscript reverse transcriptase II kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. RT-PCR was used to detect transcript levels using PCR primers as given in Table S2 and conditions as described elsewhere (Yanni et al. 2010). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control for normalization. Differences in data collected for all parameters from Wistar control and GK animals were analysed by Student's t test (SPSS version 17, Chicago, IL, USA). Data are expressed INK1197 chemical structure as the means �� SEM, and a P value selleck chemicals higher in untreated diabetic animals versus control rats. Additionally, blood glucose levels following oral glucose tolerance test were higher at all time points measured and did not return to the baseline level, indicating severely impaired glucose tolerance (P