At the end of specific treatment cells were fixed with 4% paraformaldehyde (BDH) in PBS 0.1 M pH 7.4 (Life Technologies) for 10 min at room temperature, then for another 10 minutes in 2% paraformaldehyde and washed with PBS. Samples were incubated overnight at 4 °C in PBS containing 10% normal horse serum (NHS, Thermo Fisher), 0.3% Triton X-100 (BDH), and the appropriate primary antibody. Cells' characteristics were assessed by immunocytochemistry with antibodies against Microtubule Associated Protein 2 (MAP2, polycl. 1:200; Millipore AB5622) Glial Fibrillary Acidic Protein (GFAP, polycl. 1:1000; Covance PRB571c), Nestin (monocl. 1:200; Millipore MAB353-2444141), BETA-TUBILIN III (BETA-TUB III; monocl. 1:500/20 000 GeneTex GTX631836), Chondroitin sulfate proteoglycan (NG2, 1:200; Millipore AB5320). After thorough washing with PBS and 10% NGS, cells were incubated for 90 min at room temperature with the appropriate secondary antibody (Alexa Fluor® 488 and 546, Life Technologies). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI 1µg/ml) (Sigma Aldrich), 10 minutes at room temperature, mounted using the FluorSave Reagent (Calbiochem, Merck Chemical), and analyzed by confocal microscopy (Confocal laser scanning microscopy Olympus Fluoview FV10i). The ImageJ software was used for microphotographic digital analysis. In control determinations, primary antibody was omitted and replaced with equivalent concentrations of unrelated IgG of the same subclass. The positive pixels were quantified against the negative background. The microscope light intensity of the laser was the same for all analyzed sections and for determining the background optical density.

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