The midbrain was placed in a lysis buffer containing protease inhibitors and homogenized by ultrasound on ice, centrifuged at low temperature and the supernatant was collected. BCA quantitative method was used for protein quantification. After protein quantification, the loading buffer was added to each sample and heated in a water bath at 100°C for 5 minutes to prepare the sample. The sample was separated by 10% Bis-Tris gel electrophoresis, transferred to PVDF membrane, blocked in 5% skim milk powder (w/v) for 1 h, and then incubated with anti-Tyrosine Hydroxylase (Abcam, 1:1000), anti-MHC class II (Santa Cruz, 1:500), anti-Arginase-1 antibody (Cell Signaling Technology, 1:1000) at 4°C overnight. The sample was incubated with goat anti-rabbit IgG or goat anti-mouse IgG (Beyotime, 1:1000) at room temperature for 1 h. Blots were imaged by the ECL chemiluminescence (Millipore, USA) and a Gel Image System (GE, USA). Densitometry was performed by using Image J software.
Gel image system
Manufactured by GE Healthcare
Sourced in United States
The Gel Image System is a device used for the visualization and analysis of electrophoresis gels. It captures and digitizes gel images, allowing for the documentation and interpretation of experimental results.
Automatically generated - may contain errors
Lab products found in correlation
Ecl chemiluminescence, by Merck Group (2 mentions)
Goat anti mouse igg, by Beyotime (1 mentions)
Anti tyrosine hydroxylase, by Abcam (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Anti mhc class 2, by Santa Cruz Biotechnology (1 mentions)
Anti arginase 1 antibody, by Cell Signaling Technology (1 mentions)
2 protocols using gel image system
1
Midbrain Protein Expression Analysis
2
Protein Expression Analysis in Brain Regions
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The hippocampus, cortex and midbrain (n = 6 in each group) were lysed with RIPA lysis buffer. Proteins (25 μg per lane) were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to the PVDF membrane. Each membrane was blocked in 5% skim milk powder for 1 h at room temperature and then incubated with anti-Akt (1:1,000), anti-p-Akt (1:1,000), anti-Bax (1:1,000), anti-Bcl-2 (1:1,000), anti-tyrosine hydroxylase (1:1,000), at 4°C overnight. The membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG (1:1,000) for 1.5h at room temperature. Blots were imaged by the ECL chemiluminescence (Millipore, USA) and a Gel Image System (GE, USA). Band intensities were quantified using Image J software. 2.10. Statistical analysis SPSS 22.0 software was used for data analysis. Data were expressed as the mean ± standard error of the mean (SEM). Before performing parametric tests, data distributions were tested for normality using the Kolmogorov-Smirnov test. Differences between groups in behavioral tests were analyzed using one-way ANOVA followed by Tukey post-hoc test and P < 0.05 is considered statistically significant.
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