Computational protocol: Hydrocortisone-induced parkin prevents dopaminergic cell death via CREB pathway in Parkinson’s disease model

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Protocol publication

[…] Mice were euthanized by cervical dislocation. Mouse brain subregions (VM, STR, CB) were located following procedures described previously. Dorsal posterior parietal cortex region (located above hippocampus; y coordinate from bregma = −1.7~−1.9) was dissected and used in this study and noted as CTX. Mouse brain tissues were homogenized in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-β-glycerophosphate, Phosphate Inhibitor Cocktail I and II (Sigma), and Complete Protease Inhibitor Mixture (Roche)] using a Diax 900 homogenizer. After homogenization, samples were rotated at 4 °C for 30 min for complete lysis. The homogenate was then centrifuged at 52,000 rpm for 20 min. Protein levels in the supernatants were quantified using BCA Protein Assay Kit (Pierce) with BSA as standards. Proteins were then subjected to immunoblot. Immunoblotting was performed with antibody of interest. Immuno-reactive bands were visualized with Enhanced Chemiluminescence Kit (Pierce). Densitometric analyses of protein bands were performed using ImageJ program (NIH, [...] Power analysis was performed by using G*Power 3.1 software to determine approximate sample sizes for stereological counting. On the basis of mean difference from our preliminary experiments, a total sample size of four mice was calculated to obtain significant difference (effect size f = 22.42 for 45% mean difference; α = 0.05). Data are presented as mean ± standard error of mean (SEM). Normality of the data was tested with the Shapiro-Wilk test. Statistical significance was assessed either via unpaired two-tailed Student’s t-test and nonparametric Mann-Whitney U test for two-group comparison or analysis of variance (ANOVA) test with Tukey’s honest significant difference (HSD) post hoc analysis and nonparametric Kruskal-Wallis ANOVA test for comparison of more than three groups. P values less than 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools ImageJ, G*Power
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Parkinson Disease, Fractures, Stress, Neurodegenerative Diseases
Chemicals Dopamine, Hydrocortisone