Computational protocol: Propofol Induces Apoptosis of Neurons but Not Astrocytes, Oligodendrocytes, or Neural Stem Cells in the Neonatal Mouse Hippocampus

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Protocol publication

[…] Western blot assay was performed as described previously []. Brieflly, following exposure to propofol or intralipid, brain tissues were collected and lysed and sonicated in RIPA lysis buffer (Cell Signaling, Danvers, MA, USA) containing a phosphatase and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Lysates were centrifuged at 10,000× g for 10 min at 4 °C. Total protein concentration of the supernatants was determined using a DC Protein Assay Reagents Package kit (Bio-Rad, Hercules, CA, USA). The samples were boiled for 5 min at 97 °C. Twenty five μg of protein was loaded per lane for sodium dodecyl sulfate polyacrylamide gel electrophoresis gel separation, and then transferred to nitrocellulose membrane. Membranes were blocked with blocking buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-activated caspase 3 (Cell Signaling, Danvers, MA, USA) and rabbit anti-actin (internal control; Santa Cruz, Dallas, Texas, USA). The primary antibodies were then washed out with Tris-buffered saline with 0.1% Tween 20 buffer. Subsequently, the membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling, Danvers, MA, USA) for 1 h at room temperature, and labeled proteins were detected with chemiluminescence detection reagent (Cell Signaling, Danvers, MA, USA) and obtained on X-ray film. Optical densities of activated caspase 3 and actin were quantified using ImageJ 1.47v software (Wayne Rasband, National Institutes of Health). The activated caspase 3 abundance was normalized to actin and reported as a percentage of the intralipid vehicle control. [...] All values described were expressed as the mean ± standard error of the mean. All experimental data were obtained from three mice per group. The statistically significant differences of the data between control and propofol treatment groups were analyzed by unpaired Student’s t-test using the SigmaPlot 12.5 software (Systat Software, Inc., San Jose, CA). A level of p < 0.05 was considered to be statistically significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Neurotoxicity Syndromes
Chemicals Propofol