In order to further confirm whether these cells are the downstream neural cell types, immunofluorescence was performed on differentiated primary GSCs using the specific markers of neurons and astrocytes on the 7th day in differentiating conditional medium.
Immunoblotting was performed as previously described [14] using the following specific primary antibodies: anti-filaggrin, anti-loricrin, anti-keratin 14 (Covance, Berkeley, CA); anti-HA (Sigma Chemical Corp., St Louis, MO) and anti-p21Cip1 (F-5; Santa Cruz, CA).
To verify whether downregulation of ApoE expression in our experimental conditions was associated, as expected, to increased rate of apoptosis, necrosis and inflammation, total proteins were analyzed by using the following specific primary antibodies: anti-cleaved caspase-3, anti-CREG, anti-phosphoJNK, anti-JNK, anti-phospho-p38MAPK, anti-p38MAPK, anti-NF κBp65, anti-Smad4, anti- β-actin.
For control studies, purified non-immune rabbit IgG or mouse IgG2a (Dako) were used instead of the specific primary antibodies.
For negative controls, non-immune serum was used in place of the specific primary antibody.
These included the specific primary antibody (and dilution) used for immunohistochemistry, the scoring criteria used to define positive staining and relevant clinico-pathological data.
The resulting background fluorescence was used to subtract the positive signals from using a specific primary antibody staining for the cell imaging (using the Image-Pro Plus software).
BrdU immunoreactivity was measured using a specific primary antibody provided by the kit.
For this reason, we performed western blot experiments using a specific primary antibody able to bind tau protein on the phospho-Ser396, which is recognized to be the phosphorylation site mainly related to neurofibrillary pathology in AD brain.
