Cells were washed with PBS, fixed in 4% paraformaldehyde at room temperature for 15–20 minutes and permeablized with 100% ethanol for 2 minutes at room temperature. Fixed cells were blocked in 4% BSA, 0.1% Triton X-100, in PBS at room temperature for 1 hour. Primary antibodies diluted in 0.5% BSA, 0.1% Triton X-100 in PBS and incubated overnight at 4 °C. Following incubation cells were washed 3 times for 5 minutes each in PBS then incubated with secondary antibody Alexa-fluor 488-conjugated goat anti-mouse antibodies (1:500, Invitrogen A11017), Alexa-fluor 555-conjugated goat anti-rabbit (1:500, A21430), Alexa-fluor 647-conjugated goat anti-chicken antibodies (1:500, A21449) for 1 hour at room temperature. Cells were washed 3 times in PBS and subsequently mounted onto glass slides with prolong gold anti-fade reagent with DAPI (Invitrogen, P36931). Immunofluorescence was visualized with Nikon Eclipse 80i fluorescent microscope (Nikon, Japan). The following primary antibodies were used TuJ1 (anti-β-Tubulin III; 1:500, Sigma T2200), MAP2 (1:10000, Chemicon AB5622) and GFAP (1:200, Sigma G3893) (data not shown).
