T8578

Sample cultures were fixed with 4% paraformaldehyde in PBS on ice (4 °C) for 10 min. Fixed cells were incubated with 0.2% Triton-X-100 in PBS for 5 min, then with preblock buffer (0.05% Triton-X and 5% FBS in PBS) at 4 °C for 1 h, and finally with preblock buffer containing a specific primary antibody (1:1,000) at 4 °C for 24 h. The primary antibodies used were mouse anti-L glutamate (ab9440, Abcam), rabbit anti-gamma-aminobutyric acid (GABA) (A2052, Sigma-Aldrich), rabbit anti-MAP2 (ab281588, abcom), mouse anti-β-tubulin III (T8578, Sigma-Aldrich), and rabbit anti-MBP (ab40390, Abcam), respectively. Then, the samples were incubated with the appropriate secondary antibody (anti-mouse 488 Alexa Fluor, ab150113, Abcam or anti-rabbit 546 Alexa Fluor, A11010, Lifetechnologies, 1:1,000 in preblock buffer) for 1 h at room temperature. Cell nuclei were counterstained with Cellstain DAPI solution for 1 h at room temperature. Stained cultures were washed twice with preblock buffer (5 min/wash) and rinsed twice with PBS. The immunolabeling was visualized using a confocal microscope (Eclipse Ti2-U, Nikon). Image intensity was adjusted using the ImageJ software (NIH). A Cell3 imager Estier system (Screen Holding) was used to acquire 3D images, and the images were adjusted by a Cell Visualizer software provided by the manufacturer.

After drug exposure, the sample cultures were fixed with 4% paraformaldehyde in PBS (at 4 °C) for 10 min. Fixed cells were then incubated with 0.2% Triton-X-100 in PBS for 5 min, then with preblock buffer (0.05% Triton-X and 5% FBS in PBS) at 4 °C for 1 h, and finally with preblock buffer containing a specific primary antibody, mouse anti-β-tubulin III (1:1000, T8578, Sigma–Aldrich), at 4 °C overnight. The sample cultures were then incubated with a secondary antibody, antimouse 488 Alexa Fluor (1:1000 in preblock buffer, ab150113, Abcam, Tokyo, Japan), for 1 h at room temperature. Stained cultures were washed twice with preblock buffer and rinsed twice with PBS. Local images of cell seeding area and neurite elongation area were captured by a confocal microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan). ImageJ software (NIH) was used to adjust image intensity.

The sample cultures after MEA measurements at 168 h were fixed with 4% paraformaldehyde in PBS on ice (4 °C) for 10 min. Fixed cells were incubated with 0.2% Triton-X-100 in PBS for 5 min, then with preblock buffer (0.05% Triton-X and 5% FBS in PBS) at 4 °C for 1 h, and finally with preblock buffer containing a specific primary antibody, mouse anti-β-tubulin III (1:1000, T8578, Sigma–Aldrich, St. Louis, MO, USA), at 4 °C for 24 h. The samples were then incubated with a secondary antibody, anti-mouse 488 Alexa Fluor (1:1000 in preblock buffer, ab150113, Abcam, Waltham, MA, USA), for 1 h at room temperature. Stained cultures were washed twice with preblock buffer and rinsed twice with PBS. A confocal microscope (Eclipse Ti2-U, Nikon, Tokyo, Japan) was used to capture local images, and then ImageJ software (Version 1.54g, NIH) was used to adjust image intensity.

Cells cultured on coated coverslips were washed with PBS and fixed with 4% PFA at room temperature for 15 min. Then cultures were washed three times in PBS with 0.05% Triton X-100, followed by blocking at room temperature for 30 min in TBS-Blotto (0.15 M NaCl, 20 mM Tris-HCl, pH 7.5, 4.5% non-fat dry milk) with 0.1% Triton X-100. Cells were incubated with anti Tuj1 (1:1000, T8578, Sigma-Aldrich) and GFAP antibody (1:1000, #PA1-10019, ThermoFisher Scientific) for 1 h at room temperature with shaking, washed three times, and incubated with corresponding Alexa Fluor secondary antibodies (A11029, A11012, ThermoFisher Scientific) covered with foil for 1 h. Cells were rinsed twice, stained with 300 nM DAPI for 5 min, and rinsed twice. Coverslips were mounted with mowiol and imaged using the Lionheart FX automated microscope. Coverslips were coded, and the experimentalist was blind to their assignments. In total, 15–25 fields from 3–5 coverslips were scanned under 20× objective and counted using ImageJ [29 (link)].

For confocal analysis, cells underwent our previously described double immunofluorescence protocol [9 (link)], using the following primary antibodies: (i) Rabbit polyclonal antibody against mitochondrial superoxide dismutase 2 (anti-SOD2, ab13533, Abcam); (ii) mouse monoclonal antibody to βIII-Tubulin (T8578, Sigma-Aldrich); and (iii) rabbit polyclonal antibody against Neurofilament 200 (or SMI-32, N4142, Sigma-Aldrich). Cells were then incubated with the appropriate secondary antibodies, conjugated with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA). Nuclei were stained with 1 μg/mL Hoechst (33342, Invitrogen), and slides were observed in a Leica TCS SP5 confocal microscope (Leica, Wetzlar, Germany). Random images for statistical analysis purposes were captured by Leica Application Suite software, and representative images were composed in an Adobe Photoshop CS6 format (Adobe Systems Inc., San Jose, CA, USA).