Glial fibrillary acidic protein (gfap)

Following blocking in 5% non-fat milk in tris buffered saline + Triton (TBS-T), membranes were probed overnight at 4°C with one of the following antibodies: recognizing glial fibrillary acidic protein (GFAP, 1:500, BD Pharmingen), growth associated protein 43 (GAP43, 1:5,000), ionized calcium binding adapter molecule 1 (Iba1, 1:400, Wako), myelin basic protein (MBP, 1:500, Millipore), toll-like receptor 4 (TLR4, 1:200, Santa Cruz), and tumor necrosis factor receptor 2 (TNFR2, 1:200, Santa Cruz). After extensive washes in TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000, GE Healthcare for anti-mouse and anti-rabbit, 1:1,000, Jackson ImmunoResearch for anti-rat) for 30 min at room temperature. Detection was performed with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). Quantification was performed using Quantity One software from Bio-Rad. Blots were normalized using either mouse anti-β-actin (1:500, Santa Cruz) or rabbit anti-β-actin (1:1,000, Cell Signaling).

Brain sections were fixed in 4% PFA for 15 min. After extensive washes with PBS, the sections were incubated in blocking buffer (1 % BSA in PBS containing 0.3% normal donkey serum and 0.3 % Triton X-100) for 1 h at room temperature. Next, the sections were incubated with primary antibodies [rat-anti-CD31 (1:200, BD Biosciences, 553370), mouse anti-claudin-5 (1:200, Invitrogen, USA, 35-2500), rabbit anti-ZO-1 (1:400, Thermofisher, USA, 61-7300), rabbit anti-caveolin-1 (1:500, Cell Signaling, 3238S), rabbit anti-PDGFRβ (1:200, Cell Signaling, 3169), rabbit anti-AQP4 (1:500, Millipore, USA, AB3594), rat anti-Ly6G (1:200; Biolegend, USA, 108402), rat anti-CD3 (1:200, eBioscience, USA, 14–0032-82), rat anti-CD11b (1:200, BD Biosciences, 553309), mouse anti-glial fibrillary acidic protein (GFAP, 1:200, BD Bioscience, USA, 556327), and rabbit anti-Iba1 (1:500; Wako Inc, USA, 019-19741)] overnight at 4 °C. After extensive washes, the sections were incubated with appropriate fluorescent secondary antibodies. After extensive washes, the sections were mounted with fluoromount-G with DAPI. Images were taken from peri-hematoma regions using a Nikon Eclipse Ti microscope or LSM710 confocal microscope.

Single-cell suspensions were harvested from undifferentiated or differentiated iPSCs and NT-ESCs. Undifferentiated cultures were dissociated with cell dissociation medium (CDM; Invitrogen) for 20 min at 37 °C. Differentiated cells were treated with collagenase IV for 2 h, followed by treatment with CDM for 20 min in a 37 °C incubator. The cells were filtered through a 70 μm cell strainer and incubated for 30 min at 4 °C with following anti-human antibodies: OCT4 (BD Biosciences), SSEA-3 (BD Biosciences), SSEA-4 (BD Biosciences), CD34 (Miltenyi), CD45 (BD Biosciences), E-Cadherin (BD Biosciences), SOX1 (BD Biosciences), SOX2 (BD Biosciences), NESTIN (BD Biosciences), GFAP (BD Biosciences), NKX2.1 (Abcam), CPM (Abcam), and EPCAM (Santa Cruz). For intracellular staining of OCT4, SOX1, SOX2, and NKX2.1, the cells were fixed and permeabilized using the Cytofix/Cytoperm buffer (BD Biosciences). Dead cells were excluded by 7AAD staining. Flow cytometric analysis was performed by using a FACSCanto II flow cytometer and acquired data were analyzed by the FlowJo software.

Spinal cords were frozen in Tissue-Tek OCT and cryo-sectioned into transverse 35-μm-thick slices. Slices were permeabilized for 2 hrs (3% BSA and 0.2% Triton in PBS), then incubated overnight at 4°C with primary antibodies in 3% BSA/0.2% Triton/PBS, washed in PBS and incubated 1 hr with Cy3-or FITC-linked secondary antibodies (Jackson Immuno-Research, West Grove, PA, USA). Primary antibodies include Notch1, Notch2, Notch3, doublecortin, Mash1, neurogenin2, calretinin, Olig2, NogoA, βIII tubulin (TU20; Santa Cruz Biotechnology, Dallas, TX,USA), GFAP (BD Biosciences, San Jose, CA, USA), beta-actin (Abcam, Cambridge, MA, USA) and NeuN (Millipore, Billerica, MA, USA). Antibody specificity was attested by published reports and manufacturer's data, or tested by Western blot or by showing only partial overlap with chemical detection (EdU). Slices were mounted on slides and imaged using a Nikon 80 fluorescence microscope equipped with FITC and Cy3 filters. Bleed-through was minimized by dual scanning on two different FITC-Cy3 filter sets with slightly different band pass windows. 3D imaging was performed with a Keyence BZ-9000 microscope. Stereoscopic reconstruction and quantification were carried out using NIH ImageJ. Images shown are samples of at least 10 slices stained per antibody/per condition.

The hippocampus and parenchyma were separated from the rat brains. After 3 washes, the samples were homogenized on ice into a single-cell suspension with a glass homogenizer and then centrifuged at 1200 rpm for 5 min at 4°C. The cells were resuspended and then incubated with Myelin Removal Beads II (Miltenyi Biotec, Bergisch Gladbach, Germany) to remove the myelin. After that, Fc block was used to block the cells for 30 min on ice before surface staining. Next, the cell surface was stained for 30 min on ice with 50 μl of double distilled water containing 2.5 μl of annexin V PE-cyanine 7, 5 μl of binding buffer, 0.5 μl of Zombie APC-A750 (Biolegend, CA, United States), 1 μl of CD45-BV421 and 1 μl of CD11b-BV711 (BD Biosciences, Franklin Lakes, NJ, United States). The stained cells were washed 3 times, fixed and permeabilized using fixation/permeabilization buffer (BD Biosciences, New Jersey, United States), and then probed with intracellular primary antibodies against GFAP (1:50, BD Biosciences, Franklin Lakes, NJ, United States) and NeuN (1:100, Abcam, Cam-bridge, United Kingdom) in BD Perm/Wash TM buffer. Finally, the cells were transferred into flow tubes and analyzed on a flow cytometer (Beckman Coulter, Atlanta, GA, United States), and 10000 cells per tube were captured for further analysis by CytExpert 2.0 software.