Antineuronal class 3 β tubulin

The cells were fixed by applying 4% paraformaldehyde solution for 20 min and permeabilized with 2 mg/ml BSA (Serva, Heidelberg, Germany) diluted in 0.2% Triton X-100/PBS for 10 min on ice. Nonspecific binding was blocked by 3% BSA diluted in 0.02% Triton X-100/PBS (blocking solution) for 30 min. Primary and secondary antibodies were diluted in blocking solution. The following primary antibodies, dilutions. and incubation times were used: antiacetylated α-tubulin (1/5000, T6793 monoclonal; Sigma-Aldrich), antihistone H3 acetyl k9+k14 (1/1000, 9677L; Cell Signaling), and antineuronal class III β-tubulin (802001, 1/500, overnight; BioLegend, San Diego, CA, USA). The following secondary antibodies, corresponding dilutions, and incubation times were used: donkey antimouse IgG (Alexa fluorophore-conjugated, 1/5000, 1 h; Life Technologies, Carlsbad, CA, USA) or donkey anti-rabbit IgG (Alexa fluorophore-conjugated, 1/5000, 1 h; Life Technologies). After subsequent washing with 1.5% BSA in 0.02% Triton X-100/PBS, the cover slips were mounted with 4,6-diamidino-phenylindole-containing Vectashield (Vectorlabs, Burlingame, CA, USA) to visualize the nuclei. Fluorescent micrographs were taken using a Zeiss Axio Imager M1 microscope (Carl Zeiss, Oberkochen, Germany) equipped with an AxioCam MRm3 camera (Carl Zeiss).

Cortical mixed glial cultures were prepared from P0-P2 ICR (CD-1®; Harlan Laboratories) rat pups. Neuron-glia co-cultures were maintained in complete Neurobasal medium. On Day 4 cells were infected with plenti virus containing the CMV::EPG- GFP and un-infected cells served as control.
For neuronal co-culture characterization, we used rabbit polyclonal anti- neuronal class III β-tubulin (Biolegend; 1:500 dilution) as a neuronal marker, mouse monoclonal anti-GFAP (Dako; 1:500 dilution) as a glial marker and chicken anti GFP (abcam; 1:200 dilution). After overnight incubation, the cover slips were washed thoroughly with DPBS and incubated in donkey anti mouse Alexa 405 (abcam), goat anti rabbit Alexa 594, and goat anti chicken Alexa 488 (Thermofisher Scientific), respectively at 1: 500 dilutions for 1 hour. Images were acquired using an M2 AxioImager upright epifluorescence microscope (Carl Zeiss, Oberkochen, Germany) equipped with an Apotome module and 10 × /0.45 NA and 20 × /0.8 NA objectives, and processed using AxioVision, version 4.8 (Carl Zeiss).