Image master 2 scanner

The tissue sections that were harvested were subjected to western blotting to assess protein expression following sevoflurane exposure as described previously [19 (link),20 (link)]. In brief, the brain tissue sections were carefully blended on ice using immunoprecipitation buffer (10 mM Tris– HCl of pH 7.4, 2 mM EDTA, 150 mM NaCl, and 0.5% Nonidet P-40) containing protease inhibitors (1 mg/mL leupeptin, 1 mg/mL aprotinin and 1 mg/mL pepstatin A). Protein concentrations in the collected cell lysates were determined using BCA protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amount of proteins (60 μg) were electrophoretically separated on SDS-PAGE and electro-transferred on to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies, overnight at 4°C. Following incubation, the blots were washed and further incubated with appropriate secondary antibodies. The immunoreactive bands were imagined and the images were scanned using Image Master II scanner (GE Healthcare, Milwaukee, WI, USA). The band densities of the positive bands were analysed further by ImageQuant TL software (GE Healthcare, Milwaukee, WI, USA). The expression of proteins was normalized with that of β-actin.