Computational protocol: Experimental Cancer Cachexia Changes Neuron Numbers and Peptide Levels in the Intestine: Partial Protective Effects after Dietary Supplementation with L-Glutamine

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Protocol publication

[…] Expression of ChAT, VIP, and CGRP proteins was quantified in the jejunum and ileum by Western blotting. Following laparotomy, the harvested segments were repeatedly washed with Krebs-Ringer buffer solution (pH 7.4) to completely remove the feces. The tissue was lysed by immersion in homogenization buffer (50 mM Tris HCl pH 7.2, 600 mM NaCl, 1 mM ethylenediaminetetracetic acid (EDTA) Sigma-Aldrich, Inc.) containing protease inhibitor. Lysed samples were then centrifuged for 10 min at 10,000 × g to remove the insoluble fraction. The supernatant was collected and frozen (−80°C) until further use. Supernatant total protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA, USA) []. In parallel, an equivalent fraction from each sample (30 μg) was separated by 15% SDS-PAGE (VIP and CGRP) or 12% SDS-PAGE (ChAT). The electrophoresis bands were then transferred to nitrocellulose membranes (Bio-Rad). After blocking with 5% skim milk in TBS buffer (2.24 g/L Tris base and 8 g/L NaCl, pH 7.6) with 0.1% Tween-20 for 1 h at RT, the membranes were incubated (overnight at 4°C) with primary antibodies against ChAT, VIP, and CGRP proteins (). The membranes were then incubated for 2 h at RT with a secondary peroxidase-conjugated antibody (). Secondary antibody detection employed a Novex ECL Chemiluminescent Substrate Reagent Kit (Thermo Fisher Scientific, Waltham, MA, USA), a detection system for Western blotting. Membrane chemiluminescence was detected using the ChemiDoc MP imaging system, which captures images for posterior analysis, according to the manufacturer’s instructions (V3 Western Workflow™, Bio-Rad). The bands obtained from the images corresponded to the ChAT (70 kDa), VIP (17 kDa), and CGRP (15 kDa) proteins and were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Changes in the expression of proteins analyzed by Western blotting in this study were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All protocols for electrophoresis and Western blotting were performed according to the Bio-Rad Mini Protean System standard procedures. [...] Statistical analysis was performed using Statistica 7.0 (StatSoft) and GraphPad Prism 6.0 software. (GraphPad Software)The data were presented as the mean ± standard error of the mean (SEM). Analysis of variance (two-way ANOVA) was employed for all data. The Tukey’s post-hoc test was applied whenever ANOVA indicated a significant difference. The unpaired Student’s t-test was employed for comparisons between two groups. The significance level was defined as p < 0.05. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Gastrointestinal Diseases, Neoplasms
Chemicals Glutathione