Anti nestin

U87MG cells or GSCs were treated with DMSO (control), NHI-1 or NHI-2 (10 μM) for 48 h or 7 days, respectively. At the end of the treatment periods, the cells were collected and lysed. Equal amounts of the cell extracts (40 μg of protein) were diluted in Laemmli sample solution, resolved using SDS-PAGE, transferred to PVDF membranes and probed overnight at 4 °C using the following primary antibodies: anti-nestin (sc-20978, Santa Cruz Biotechnology, Heidelberg, Germany; 1:150); anti-GFAP (sc-9065, Santa Cruz Biotechnology; 1:50); anti-NeuN (ab177487, Abcam, Cambridge, UK); and β-actin (MAB1501, Merck KGaA, Darmstadt, Germany)). The primary antibodies were detected using the appropriate peroxidase-conjugated secondary antibodies, which were then detected using a chemioluminescent substrate (ECL, Perkin Elmer). Densitometric analysis of the immunoreactive bands was performed using Image J Software.

Seven, seven and six animals of both MSC treatment and control groups were sacrificed at days 3, 7, 14 respectively. Brain tissue of all animals was removed, fixed in 10% and was processed for paraffin-embedded sections for histological examinations and immunostaining evaluation. The MSC trafficking was studied with immunohistochemistry using anti-GFP (Abcam). For the determination of in vivo differentiation, MSCs from wild-type SD rats were pre-labeled with CM-DIL (Life Technologies) and the co-expression of red fluorescence with FITC conjugated anti-GFAP, anti-Nestin (Santa Cruz) and anti-NeuN (Millipore) was examined using immunofluorescence staining. Apoptosis staining was done according to manufacturer’s instructions (Roche Diagnostics). Immunohistochemistry staining on paraffin sections using anti-GFP, anti-GFAP, anti-Iba1 (Abcam), PCNA (Wako) was performed according to standard procedures. The neuronal death was studied with Cresyl Violet staining. The expression of chemokine receptor, CXCR4 and stromal SDF-1 (Abcam) were studied using immunofluorescent staining on paraffin sections.

Total cellular proteins and nuclear proteins were separately extracted using the Total Protein Isolation Kit (Sangon Company, China) and Nuclear Protein Isolation Kit (Sangon Company). Proteins were quantified by Bicinchoninic acid assay kit (Sangon Company). The protein samples were loaded on SDS–PAGE gels and electrophoresed under standard conditions. Western blotting was performed using nitrocellulose membranes. After blocking, membranes were incubated with primary antibodies (1:200–1:500) at 4 °C overnight. After rinsing, incubation was conducted with secondary horseradish peroxidase-conjugated goat anti-rabbit or mouse IgG antibody (Bioss Biotechnology, China) and exposed to film. Primary antibodies included anti-Frizzled (FZD, Immunoway, Catalog: YT1783, USA), anti-phospho-Frizzled (p-FZD, Immunoway, Catalog: YP0173), anti-β-Catenin (Boster Biotechnology, Catalog: BM1575, China), anti-Oct4 (Santa Cruz Biotechnology, Catalog: sc-5279, USA), anti-Sox2 (Santa Cruz Biotechnology, Catalog: sc-17320), anti-βIII-Tubulin (Santa Cruz Biotechnology, Catalog: sc-58888), anti-Map2 (Santa Cruz Biotechnology, Catalog: sc-20172), anti-Nestin (Santa Cruz Biotechnology, Catalog: sc-20978), and anti-Gapdh (Cell Signaling Technology, Catalog: #2118, USA).

Total protein extracts (50 μg) of U87MG GSCs and C6 GSCs treated with FK506 and/or Vc were separated by 8% and 15% SDS-PAGE, followed by the transfer to 0.22-μm PVDF (polyvinylidene fluoride) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked using 5% non-fat milk for 45 min and then incubated with anti-CD44 (#3570, Cell Signaling Technology, Inc., Beverly, MA, United States), anti-nestin (sc-33677, Santa Cruz Biotechnology, Dallas, Texas, United States), anti-cleaved caspase-3 (#9661, Cell Signaling Technology, Inc., Beverly, MA, United States), anti-Bcl-2 (MAB8272, R&D Systems, Inc. Minneapolis, United States ), and anti-Bad (MAB6405, R&D Systems, Inc. Minneapolis, United Stated) antibodies overnight. Finally, membranes were incubated with a secondary HRP (Horseradish Peroxidase)-conjugated IgG antibody (DAKO Agilent, Santa Clara, CA, United States) for 1 h at room temperature, and detected using SuperSignal^TM West Dura (Thermo Fisher Scientific Inc., Waltham, MA, United States) in the image analysis system syngene G: Box (Synoptics Ltd., Cambridge, UK). The images were analyzed by densitometry (Image J software, 1.52a, NIH, Bethesda, MD, United States) and normalized by β-actin (sc-47778-HRP, Santa Cruz Biotechnology) expression using vehicle-treated (1× PBS) cells as a calibrator.

For immunocytochemical studies, cells derived from NLBs were seeded onto coverslips fibronectin-poly D lysine coated and fixed with 4% (wt/vol) paraformaldehyde in 0.1 M PBS. Then, they were incubated for one hour in 2.5% (wt/vol) bovine serum albumin (BSA) in PBS and with primary and secondary antibodies. After washing, the coverslips were mounted on slides with Fluoro-Gel (EMS, Hatfield, PA, USA) and fluorescent signals were detected using a Leica Spectra confocal microscope. The primary antibodies used were anti-PAX6 (rabbit polyclonal, 1:20) and anti-NESTIN (goat polyclonal, 1:400) (Santa Cruz Biotechnology, Inc). The secondary antibodies used were anti-rabbit IgG labeled with Cy2 (1:200) and anti-goat IgG labeled with Cy5 (1:200) (Jackson Immuno Research Laboratories, Inc.). The nuclei of cells were counterstained with DAPI. For the quantification, ten images were taken per condition in which at least a number of 700 cells were counted.

Six weeks after the implantation, the animals’ spinal cords were isolated. A 1-cm fragment was collected, including the epicenter of the lesion and equal rostral end caudal portions. The non-degraded PLGA membranes were removed and the tissue was mechanically and enzymatically dissociated with trypsin (Sigma-Aldrich). The cell suspension was fixed for 30 min with 4% paraformaldehyde and subsequently blocked for 30 min with 3% BSA in PBST. After blocking for 20 min with 3% BSA in PBS with 0.1% Triton X-100, the cells were incubated with primary antibodies, including anti-GFAP (DAKO; 14.5 μg/mL), anti-βIII tubulin (Millipore, 05559), anti-nestin (Santa Cruz, SC-33677, 1 μg/mL), and anti-CD68 (Millipore, MAB 1435). The cells were washed twice with PBS1X and incubated for 1 h with the secondary antibody Alexa-fluor 488 anti-mouse or anti-rabbit (10 μg/mL, Thermo Fisher Scientific, USA) at 37°C. Negative controls (samples incubated only with the secondary antibody) were included for setting up the machine voltages and to determine the negative population. The cells were analyzed using a FACSAria III cytometer (Becton Dickinson Biosciences, USA), equipped with a 488 nm argon laser and the FACSDiva 6.0 software. An average of 5×10⁴ events was analyzed.