Odyssey image scanner

Pre-cooled PBS was added to the 6-well plate, and the adherent chondrocytes were washed by gently shaking 3 times, and then RIPA buffer containing 10% phosphatase inhibitor and protease inhibitor (Roche Diagnostics, Basel, Switzerland) was added to each well; after that total protein was extracted from the chondrocytes. Subsequently, we measured protein concentration, utilizing the experimental tool BCA assay kit. SDS-PAGE gels was used to separate equal amounts of protein (15–20 μg), and then transferred onto polyvinylidene fluoride (PVDF) membrane measuring 0.22 µm. After that, the PVDF membrane was placed in the configured 5% non-fat milk, and then incubated on a horizontal shaker with shaking. After sealing was completed, the blocked membrane was placed in TBST and then allowed to be washed by shaking on a horizontal shaker for a total of three times. The membranes were then incubated sequentially with primary antibody (4°C, overnight) and secondary antibody (1 h). Finally, the membrane was detected employing the Odyssey image scanner (Li-COR. Inc., Lincoln, NE, United States). ImageJ was used to quantify the gray value of blots.

For Western blot studies, rats were killed by decapitation following anesthesia with halothane, and hippocampi were rapidly dissected and homogenized in cold lysis buffer (50 mm Tris- HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 1 mm EGTA, 1 mm PMSF, 10 mm NaF, and 1 mm Na3VO4), containing protease inhibitor cocktail (Thermo Scientific). Fifteen to twenty micrograms of proteins were loaded onto 8-15% SDS gels, separated by gel electrophoresis, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% Odyssey blocking buffer for 1 h at room temperature, and incubated overnight in PTEN and drebrin primary antibodies at 4°C. After washing to remove free primary antibodies, membranes were incubated in fluorescent secondary antibodies for 30 min at 20°C. After a final wash, membranes were scanned by Odyssey image scanner (LI-COR Biosciences). Blots were stripped and reprobed with an antibody against actin, and the levels of PTEN and drebrin were normalized to those of actin. Results were averaged and normalized to the mean values in the control group.