Mouse anti tuj1

Total protein was extracted with cell lysis buffer (Solarbio, Beijing, China). Proteins were separated by 10% SDS‐PAGE and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 5% non‐fat milk (Sangon Biotech, Shanghai, China) for 2 h, then incubated with primary antibodies overnight at 4°C, and finally incubated with HRP‐conjugated secondary antibodies for 2 h at room temperature. Next, immunoreactive bands were detected using an enhanced chemiluminescence kit (Bio‐Rad, Hercules, CA, USA). β‐Actin was used as a loading control. The primary antibodies included mouse anti‐MAP2 (Millipore; 1:1000), mouse anti‐Tuj1 (Millipore; 1:1000), rabbit anti‐GFAP (Millipore, 1:1000), rabbit anti‐Acsl4 (Abcam; 1:1000), rabbit anti‐Akt (Cell Signaling Technology, Danvers, MA, USA; 1:1000), rabbit anti‐phospho‐Akt (Cell Signaling Technology; 1:1,000), rabbit anti‐PI3K (Abcam; 1:1000) and mouse anti‐β‐actin (Abcam; 1:1000).

Human NSCs and differentiated neuronal cells were seeded on chamber slides. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 in TBS for 5 min at room temperature. TBS with 2% FBS was used for blocking. The cells were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-SOX2 (1:200, Cell Signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-Tuj1 (anti-neuron-specific class III β-tubulin, 1:200, Millipore, MA, USA), mouse anti-GAD67 (1:200, Millipore, MA, USA), rabbit anti-active caspase 3 (1:200, Millipore, MA, USA), rabbit anti-LC3A/B (1:200, Cell Signaling, CA, USA), mouse anti-EV-A71 3D (1:500, Genetex, CA, USA) or rabbit anti-EV-A71 3A (1:500). The cells were then washed with TBS and incubated with the following secondary antibodies for 1 h at room temperature: DyLight 488-conjugated goat anti-rabbit secondary antibody or DyLight 594-conjugated donkey anti-mouse secondary antibody (1:1,000, Jackson ImmunoResearch Laboratories, Pennsylvania, USA). The cells were then washed with TBS, and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA). Images were collected with an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) and an LSM 510 microscope (Zeiss, Jena, Germany).

The following antibodies were used: mouse anti-Tuj1 (1:50, Millipore), mouse anti-microtubule-associated protein (MAP) 2 (1:200; Sigma–Aldrich), rabbit anti-GFAP (1:200; Millipore), mouse anti-O4 (1:100, Millipore), mouse anti-CNPase (1:100, Millipore), goat anti-Sox2 (1:100, Santa Cruz), mouse anti-Nestin (1:100; Developmental Studies Hybridoma Bank, Beijing, China), mouse anti-Pax6 (1:100; Developmental Studies Hybridoma Bank), donkey Alexa-555 anti-mouse (1:1000; Invitrogen), and donkey Alexa-555 anti-rabbit (1:1000; Invitrogen). Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The slide-mounted, stained samples were observed using an LSM 510 META confocal microscope (Zeiss, Oberkochen, Germany) with excitation wavelengths of 543, 488, and 405 nm. The channel signals were collected sequentially. Collected images were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA).

Cell monolayers and NSPs were fixed in 4% PFA for 20 minutes at 4°C and then washed briefly in PBS. NSPs were embedded in Tissue-Tek OCT compound (Labtek) and cut at 15 μm on a cryostat, and sections were placed on superfrost slides. After washing in PBS, sections or culture dishes were permeabilized with 0.1% Triton-X-100 in PBS (PBT) for 5 minutes and then blocked in 10% fetal calf serum in PBT for 60 minutes at room temperature. Samples were then incubated with primary antibody (diluted in the block buffer) overnight at 4°C. The following primary antibodies were used: goat anti-SOX2 (1 : 200, R&D Systems), mouse anti-PAX6 (1 : 80, DSHB), rabbit anti-TBR2 (1 : 1000, Chemicon), mouse anti-TUJ1 (1 : 1000, Millipore), rabbit anti-KI67 (1 : 600, Abcam), and mouse anti-S100β (1 : 500, Sigma). Following three 5 minute washes in PBT, ALEXA Fluor secondary antibodies (Life Technologies/Invitrogen) (1 : 1000 diluted in the block buffer) were applied for 1 hour at room temperature. All samples were counterstained with 49,6-diamidino-2-phenylindole (DAPI; 1 μg/ml, Sigma-Aldrich). Samples were then mounted onto glass slides with 5 μl of moviol aqueous mountant followed by viewing and image capturing under Zeiss Axio Observer z1 fluorescence microscope using ZEN imaging software.

To stain with various antibodies, brain sections were incubated for 1 h at room temperature in blocking solution (10% FBS, 0.3% Triton X-100), then in the primary antibody at 4°C overnight. Sections were washed 3×20 min in 0.3% Triton X-100 in PBS, then incubated in Alexa-Fluor-conjugated or DyLight-conjugated secondary antibodies for 2 h at room temperature. Incubation with DAPI for 5 min and 3×20 min 0.1% Triton X-100 washes followed, both at room temperature.
Primary antibodies used in this study were: rabbit polyclonal anti-PRDM16 (1:500; Bjork et al., 2010 (link)) and goat anti-SOX2 (1:150; R&D Systems), mouse anti-TUJ1 (1:1000; Millipore), rabbit anti-Ki67 (1:500; Abcam) and rabbit anti-Caspase-3 (1:200; Cell Signaling). The secondary antibodies used were: donkey anti-rabbit-647 (1:1000; Jackson ImmunoResearch), donkey anti-goat-549 (1:1000; ImmunoResearch) and donkey anti-mouse-647 (1:1000; Jackson ImmunoResearch). Imaging was done on a Leica CTR6500 confocal laser-scanning microscope. Volocity (ImproVision) and Photoshop (Adobe Systems) softwares were used for image processing.

Cells were rinsed with ice-cold PBS, fixed in 4% PFA for 30 min at 4 °C, washed three times with PBS, permeabilized for 10 min at 4 °C in PBS plus 0.1% Triton X-100 (by volume), blocked in PBS with 5% BSA for 30 min at 4 °C, and washed three times in PBS. The primary antibodies used were rabbit anti-Aldhl1l (1:1000, Abcam), rabbit anti-Aqp4 (1:300, SantaCruz); rat anti-active β1-integrin (9EG7) (1:50, BD Bioscience) rat anti-GFAP (1:2000, Invitrogen), rabbit anti-P-smad1/5/8 (1:1000, Cell Signaling), rabbit anti-Tuj-1 (1:600, Abcam), mouse anti-Tuj-1 (1:100, Millipore), and secondary antibodies used included donkey antibodies to rat, rabbit and mouse conjugated with Alexa Fluor 488, 594 or FITC (1:200, Jackson ImmunoResearch Laboratories).