Imagequant las 500

For the western blotting analysis, 30 μg of the total cell lysis was separated using 12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Merck-Millipore, Germany) with a 0.22 μm aperture. After blocking with 5% bovine albumin dissolved in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween-20, pH 8.0), the membrane was washed three times with TBST. Then, the membrane was incubated with primary antibody at a 1:1000 dilution overnight. The membrane was washed three times with TBST for three times, and then incubated with HRP conjugated secondary antibody at a 1:5000 dilution for two hours in room temperature. The membrane was washed three times with TBST, and then washed three times with TBS. The target protein bands were detected using ImageQuant LAS 500 (GE Healthcare, USA). The primary antibodies and secondary antibodies used in this study were purchased from Cell Signaling Technology (Cell Signaling Technology, USA).
For the biotin-streptavidin blotting analysis, the protein samples separating and transferring steps were the same as western blotting, but the membrane was incubated with HRP labeled streptavidin at a 1:500 dilution for at least 1 hour at room temperature. The membrane was then washed three times with TBST, and then washed three times with TBS. The target protein bands were detected using ImageQuant LAS 500 (GE Healthcare, USA).

RT-PCR bands were captured and digitalized under UV light using ImageQuant LAS 500 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The optical density of the bands for Ten-2, TCAP-2, GADPH and NFL gene expressions were analyzed by densitometry using ImageQuant LAS 500 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) software. Absolute data were normalized with GADPH and expressed in relation to NFL gene expression. Ten-2 data were expressed in relation to NFL gene expression, as Ten-2 is present in cortical neurons in control animals and in neurons and astrocytes in animals with cerebral cortex injury. This analysis is more confident because we collected samples more restricted to the injury area, reducing the healthy tissue around it to the maximum, where NFL expression is more elevated due to presence of more neurons. Thus, we can infer that if there is some increase in the Ten-2 gene expression in samples from animals with cerebral cortex injury, this expression increase most likely comes from Ten-2 reactive astrocyte gene expression than from neurons, since NFL expression is low in these animals group.
The gene expression data had normal distribution and were submitted to ordinary one-way ANOVA, followed by Dunnett's multiple comparison post-hoc tests, considering p < 0.05 as significant (GraphPad Prism 6, GraphPad Software, Inc., CA, USA).

For determining p-AKT level relative to total AKT, Western blot technique was used. Briefly, cellular proteins of the untreated and treated spheres were extracted, quantified by protein assay dye reagent concentrate, boiled in loading buffer, and separated using 4%–20% Mini-PROTEAN TGX™ gel. Then, proteins were transferred to nitrocellulose membrane, blocked with 5% bovine serum albumin, washed, and incubated with 1:1,000 p-AKT (rabbit monoclonal antibody Thr 308) or AKT overnight at 4°C. After that, the membrane was washed, incubated 1: 2000 secondary antibody (anti-rabbit IgG) for 1 h, washed, and chemiluminescent substrate was added, then bands were documented using chemiluminescent imaging system (ImageQuant™ LAS 500, GE Healthcare Bio-Sciences, Piscataway, NJ, US). For housekeeping protein, the membrane was washed, and incubated with 1:1000 HRP-β-actin for 1 h, washed, and the substrate was added for visualizing its bands using chemiluminescent imaging system (ImageQuant™ LAS 500, GE Healthcare Bio-Sciences, Piscataway, NJ, US).