Rabbit anti map2

Cells, including human ADSCs, neurospheres, and the terminal differentiated cells of neurospheres, were subjected to immunocytochemistry. Cells were seeded on poly-l-lysine-coated cover slips, fixed with 4% paraformaldehyde for 30 min, permeabilized using 0.25% Triton X-100 (Amresco, diluted in PBS) for 18 min, incubated with 5% bovine serum albumin (Solarbio, diluted in PBS) for 60 min at room temperature, and reacted overnight at 4°C with the following antibodies: rabbit-anti-Nestin (1 : 150 diluted in PBS; Sigma), rabbit-anti-GFAP (1 : 1000 diluted in PBS; Abcam), rabbit-anti-MAP-2 (1 : 50 diluted in PBS; Abcam), and mouse-anti-NeuN (1 : 50 diluted in PBS; Milipore). Then, cells were incubated for 60 min with secondary antibody, either Cy3-conjugated goat anti-rabbit (1 : 100 diluted in PBS; Peprotech) or TRITC-conjugated goat anti-mouse (1 : 100 diluted in PBS; Peprotech), and were labeled with 4′,6-Diamidino-2-phenylindole dihydrochloride (1 : 100 diluted in PBS; Sigma) for 5 min. Immunostaining patterns were visualized with a fluoromicroscope (Olympus).
For cell proliferation studies, human ADSCs and neurospheres were stained with rabbit anti-Ki-67 (1 : 1000 diluted in PBS; Abcam) according to the procedure described above.

Free-floating brain sections were prepared in the same way as for immunohistochemistry. Sections were blocked in PBST containing 5% normal goat serum for 30 min at room temperature, and incubated with both mouse anti-tau CP27 (a human specific tau antibody) (1:1000) and rabbit anti-MAP2 (a specific neuronal marker) (Abcam; 1:1000), rabbit anti-IBA-1 (1:500) or rabbit anti-GFAP (1:5000) antibodies in PBST containing 5% normal goat serum at 4°C overnight. For IBA-1 labeling, 10 μg/ml rat anti-mouse FcR block (BD Biosciences) was added at the block stage. After three washes with PBST, sections were incubated with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies; 1:500) for 1 h at room temperature on a rotator. Following three washes with PBS, autofluorescence in sections were quenched with 0.3% Sudan black in 70% ethanol (Decon Laboratories, King of Prussia, PA, USA) for 6 min at room temperature. The sections were rinsed with 70% ethanol and washed three times with 0.02% Tween-20 in PBS [25 (link)]. The nuclei were stained with 5 μg/ml Hoechst33342 (Sigma-Aldrich) in PBST for 10 min at room temperature. Following three washes with PBS, sections were mounted on slides using SlowFade gold anti-fade reagent and imaged using confocal laser scanning microscopy via Z-stack.

After FA challenge, the plates were placed on ice, media was removed and the cells were fixed with ice-cold 4% paraformaldehyde for 20 min, washed in PBS (3 X 5 min) then permeabilised with 0.2% Triton X-100 for 15 min. Cells were washed 3 times with PBS. Non-specific staining was blocked with 8% BSA dissolved in PBS for 20 min. Cells were incubated in primary antibody rabbit anti-MAP2 (Abcam, UK) at [1:1000] for 1 h followed by 3 washes in PBS. Cells were then incubated in secondary antibody goat anti-rabbit Alexa Fluor 488 (Molecular Probes, USA) at [1:1000] for 1 h. Both primary and secondary antibodies were diluted in PBS containing 4% BSA and 0.2% Triton X-100. Control slides omitted the primary antibody which resulted in a complete lack of staining (data not shown). Coverslips were mounted onto glass slides using Vectashield (Vector Laboratories, UK). Cells were imaged using a Leica DMR microscope fitted with a QImaging QICAM FAST 1394 digital camera.

Cortices were microdissected from E18.5 embryos, digested in trypsin (Pierce), manually triturated and plated on 35-mm well onto poly-D-lysine coated coverslips at a density of 150,000 cells for the neurite outgrowth assay or 500,000 for cellular localization experiments. Cells were plated in medium containing DMEM with 10% FBS and 1% penicillin/streptomycin and 4 h after plating, media was completely removed and replaced with Neurobasal media containing B-27+ (Gibco/Invitrogen), 1% penicillin/streptomycin, and L-glutamine. Cells were cultured for 24 h or one week in a 37°C incubator containing 5% CO₂, then fixed for 10 min in 4% paraformaldehyde. Immunocytochemistry was conducted using the following antibodies: mouse anti-Tau (1:200; Abcam), rabbit anti-MAP2 (1:1000; Abcam), rabbit anti-Mllt11 (1:300, Abcam), rabbit anti-Tuj1 (Tubb3; 1:1000, Biolegend), and mouse anti-acetylated α-tubulin (1:1000, Sigma).

Honokiol (HY-N0003), MK-801 (HY-15084B) and Compound C (HY-13418A) were purchased from MCE (Shanghai, China). BAPTA-AM was purchased from Thermo Fisher Scientific (Cat# B6769); cantharidin was purchased from Institute for Drug Control (Shanghai, China). All primary antibodies used in this study included: Rabbit anti-NMDAR2B (Abcam, Cambridge, UK); Fluorescein isothiocyanate (FITC) anti-SIRT3 (Biorbyt, Cambridge, UK); Rabbit anti-PGC-1α (Santa Cruz, CA, USA); Rabbit anti-AMPKα and Rabbit anti-phospho-AMPKα (Thr172) (Cell Signaling Technology, MA, USA); Rabbit anti-MAP2 (Abcam, Cambridge, UK); Mouse anti-NeuN (Abcam, Cambridge, UK); Rabbit anti- CaMKKβ and Rabbit anti-Phospho-CaMKKβ (Abcam, Cambridge, UK); Phosphatase 4 (Santa Cruz, CA, USA); Rabbit anti-GAPDH and Rabbit anti—beta Actin (Servicebio, WuHan, China).