Computational protocol: Testosterone Induces Molecular Changes in Dopamine Signaling Pathway Molecules in the Adolescent Male Rat Nigrostriatal Pathway

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Protocol publication

[…] Dorsal striatum or substantia nigra tissue blocks were homogenized (0.1 M Tris, pH 7.5, 50% glycerol, proteinase inhibitor cocktail (Sigma Cat# P8340) and aprotinin 0.015 mM, Sigma) using a handheld electric homogenizer (Polytron, Kinematica, Lucerne, Switzerland). Protein concentration was determined using the Bradford protein assay (Sigma). An aliquot of each sample was combined and used as a standard and run in duplicate on each gel to allow standardization between blots (internal control). Standard curves with between 0.5 and 20 µg substantia nigra or dorsal striatum protein were run and TH and DAT expression was determined to be within a linear range and 3 µg protein/sample was used. SDS-PAGE (10% acrylamide for TH and 8% acrylamide for DAT) was performed and proteins were transferred to nitrocellulose (45 µm, Biorad, CA, USA). Primary antibodies were anti-TH (host species rabbit, 1∶5000; Chemicon), anti-DAT (host species rabbit, 1∶300, H-20 sc14002, Santa Cruz) and anti-β-actin (host species mouse, 1∶5000; MAB1501, Millipore). Secondary antibodies were goat anti-mouse or anti-rabbit horseradish peroxidase conjugated (1∶4000, Millipore). Immunoreactive bands were detected using the LumiGlo detection kit (LumiGlo Reagent; Cell Signaling, Danvers, MA, USA) on hyperfilm (Amersham, GE Healthcare, Uppsala, Sweden). The immunoblots were scanned and band densities converted to numerical values using ImageJ software (ImageJ 1.43u, National Institutes of Health, USA). Relative intensities of TH and DAT bands (on separate blots) were normalized to relative intensity of β-actin bands on the same immunoblot. [...] Unless otherwise stated statistical analyses were conducted using SPSS software (IBM SPSS Statistics, version 19) and p<0.05 was considered statistically significant. Population outliers were removed by Grubb's test (GraphPad Prism online calculators) on the normalized qPCR, immunoblotting and HPLC data. Immunoblotting data are presented as change of relative intensity compared to the Gdx group ± SEM. Immunoblotting data were normalized to β-actin expression. qPCR data are presented as percent change of mRNA levels relative to the Gdx group ± SEM. Outlier detection of the triplicates obtained from the qPCR raw data excluded measurement errors . qPCR raw data was normalized by the geomean of the housekeepers. HPLC data is expressed as ng/mg tissue ± SEM. HPLC raw data was normalized to an internal control run each day. Dopamine turnover was calculated before outliers were removed. All data was analyzed by one-way ANOVA followed by Fisher's LSD. Comparisons of DAT protein levels were made using one-directional t tests (GraphPad Prism) due to an a priori hypothesis , based on mRNA findings, that DAT protein would be increased by androgens relative to the Intact and Gdx groups. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Rattus norvegicus
Chemicals Androgens, Dopamine, Estradiol, Dihydrotestosterone, Testosterone