Computational protocol: Neuronal Expression of the Human Neuropeptide S Receptor NPSR1 Identifies NPS-Induced Calcium Signaling Pathways

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Protocol publication

[…] The complete eluent from the agarose beads (membrane fraction) and 29% of the supernatant (unlabeled intracellular proteins) were separated in a 10% SDS-PAGE and subsequently blotted on nitrocellulose membranes (Perkin Elmer) in transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3). Membranes were blocked for 1h at RT in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5) containing 4% non-fat dry milk (Fluka). Membranes were incubated with primary antibodies (rabbit anti-mCherry, 1 mg/ml, 1:1,000, ab167453, Abcam and rabbit anti-neuronal class III β-tubulin, 1 mg/ml, 1: 40,000, PRB-435P, Covance) at 4°C overnight in blocking buffer. After washing, the secondary antibody (horseradish peroxidase-coupled goat anti-rabbit immunglobulins, 1:2,000, DAKO) was applied for 1h at RT in TBS-T/4% milk. After washing, immunosignals were detected using a supersignal western blot detection kit (Thermo Scientific) and a Chemidoc MP imaging system (Biorad). Exposure time was 4 s for the biotinylated fraction and 0.45 s for the unlabeled fraction. Immunoblots were analysed using the software ImageJ []. [...] The software ImageJ [] and Excel 2010 (Microsoft, USA) was used for analyzing calcium imaging data. ROIs were applied to the soma of all glutamate-responsive neurons, defined by an increase of fluo-4 fluorescence intensity of the soma of at least 5x over baseline upon glutamate application. Intensity values from individual ROIs were normalized to baseline conditions prior to substance application. Calcium signals of individual cells were peak-aligned using Clampfit10 (Molecular Devices Corporation, USA). Fluorescence traces were averaged and plotted with Origin 9.0 (OriginLab, USA). Fluorescence amplitudes were calculated referring to baseline conditions. Amplitude values for baseline conditions represent maximum values within the baseline referring to the mean values of the baseline. Integration of the response amplitude over time for slow component of the calcium signal was done in Clampfit10. Image sequences from immunocytochemistry were volume rendered with EZ-C1 Viewer (Nikon). Prism (Graphpad, USA) was used for statistical analysis. One-way ANOVA with Tukey post-hoc tests or student’s t-tests were used as applicable. P values for Tukey post-hoc tests are given as multiplicity adjusted p values. Asterisks in figures indicate significant differences in the tested datasets (* = p < 0.05, ** = p < 0.01, *** = p < 0.001). Data are presented as mean with standard deviation if not indicated otherwise. All figures were prepared for presentation using CorelDraw Graphics Suite 12 (Corel Corporation, USA). […]

Pipeline specifications

Software tools ImageJ, CorelDraw
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Diseases Protein S Deficiency
Chemicals Calcium, Ryanodine