Ab177487

Tissue sections or adherent cultured cells were incubated for one hour at room temperature in blocking solution (50 mmol/L Tris-HCl, pH 8.0, 0.1 mol/L NaCl, 0.1% Triton X-100, 3% NGS, 0.1% BSA) prior to overnight primary antibody incubation (4°C). Primary antibodies for immunofluorescence studies were: SOX2 (1:1000 dilution, Ab5603, Millipore), Ki67 (1:500 dilution, ab15580, Abcam), BrdU (1:500 dilution, ab6326, Abcam), NeuN (1:500 dilution, ab177487, Abcam), TUJ1 (1:200 dilution, T8660, Sigma-Aldrich), GFAP (1:500 dilution, Z0334, Agilent), MBP (1:300 dilution, SMI-99P, Covance), OLIG2 (1:200 dilution, AB9610, Millipore), OLIG2 (1:50 dilution, MABN50, Millipore), IBA1 (1:200 dilution, ab5076, Abcam), CD31 (1:100 dilution, ab24590, Abcam), ACTA2 (1:400 dilution, ab5694, Abcam), GFP (1:2000 dilution, ab13970, Abcam), CD31 (1:300 dilution, AF3628-SP, R&D), CD146 (1:100 dilution, 134701, BioLegend). We used immunofluorescence staining with Alexa Fluor 488, 555, or 647 (Life Technologies) and biotin-streptavidin-Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch), as well as horseradish peroxidase-based Vectastain ABC Kit (Vector Laboratories). Image acquisitions were performed using a Zeiss LSM880 confocal microscope and image editing done using ZEN, Photoshop or ImageJ.

Animals were deeply anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and underwent sternotomy, followed by intracardiac perfusion with 200 ml saline and 200 ml 4% ice-cold paraformaldehyde in 0.1 M phosphate-buffered saline. The brain was removed, postfixed in 4% paraformaldehyde for 4 h, and subsequently allowed to equilibrate in 30% sucrose in phosphate-buffered saline overnight at 4°C. Brain samples were embedded in Tissue-Tek O.C.T., cut at a thickness of 20 μM using a cryostat (Leica CM1850-1-1), and mounted on slides. All Brain samples were thoroughly rinsed in PBS to remove any residual OCT. The samples were then blocked in 0.3% Triton-X 100 and 5% normal donkey serum (NDS) in PBS for 1 h, and incubated overnight with anti-MHCII (1:50, MCA46GA, BIO-RAD), anti-IBA-1 (1:100, ab178847, Abcam), anti-GFAP (1:100, ab254082, Abcam), anti-NEUN (1:100, ab177487, Abcam). On the next day, the samples were rinsed in PBS and incubated for 1 h in PBS containing appropriate secondary antibody (1:500) from Jackson ImmunoResearch (catalog nos. 711-545-152 or 715-585-150) or Cell Signaling Technology (catalog nos. 8,889 or 4,408). The samples were then rinsed in PBS and coverslipped with Antifade Mounting Medium with DAPI (Beyotime, Nanjing, China). The imaging and subsequent analysis were performed using a Confocal Microscope (TCS SP8, Leica Microsystems, Mannheim, Germany).

Brain tissues (n = 6) were put into 30% sucrose solution for 24 h and cut into 30 μm coronal sections using a Leica CM1950 cryostat (Leica Microsystems). After washing with PBS, coronal sections were sealed with 10% goat serum (C0265, Beyotime) and then incubated with primary antibodies of Drp1 (ab184247, Abcam) and Parkin (JF82-09, Novus). The sections were then sealed with 10% goat serum for 1 h and then incubated with Tomm20 antibody (H00009804-M01, Abnova). Thereafter, the sections were stained with fluorochrome-conjugated secondary antibody (ZF-0311, ZSGB-BIO), glial fibrillary acidic protein antibody (1 : 5000; ab7260, Abcam), and ionized calcium-binding adapter molecule 1 antibody (1 : 500; 019-19741, Wako). In brief, paraffin sections were immunostained with anti-nuclei (NeuN) antibody (ab177487, Abcam) at 4°C overnight and subsequently subjected to terminal deoxynucleotidyl transferase dUTP nick-end labeling staining using an In Situ Cell Death Detection kit (Cat. No. 11 684 795 910) according to the manufacturer's protocol. Finally, the sections were incubated with 4′,6-diamidino-2-phenylindole (C1002, Beyotime) nuclear dye, and the sections were visualized using a confocal laser scanning microscope (FV1000, Olympus). Sections from forebrain, midbrain, and hindbrain regions were randomly selected, and the ischemic penumbra was counted for statistical analysis.

Pups were sacrificed at E14.5 and the embryonic brains were fixed, frozen, and cut into sagittal sections as previously described. An antigen retrieval step was performed by incubating the slides with 10mM Sodium Citrate buffer 10 min at 100 °C. Slides were then allowed to cool down and rinsed once with PBS before blocking 1h at room temperature with 10%NGS and 0.1% Triton X-100 in PBS. Anti-PH3 antibody (06-570, Merck Millipore) and anti-NeuN (ab177487, Abcam) were used alone while anti-PAX6 (901301, Biolegend) and anti-TBR2 (14-4875-80, ThermoFisher) antibodies were used concomitantly for co-staining. In all conditions, primary antibody was incubated overnight at 4 °C. After three washes with 0.1% Triton X-100 in PBS, slides were incubated with a matching secondary antibody (Alexa fluors, Thermo Fisher). Slides were then washed three times in PBS and mounted and scanned as previously described. We counted the PH3⁺ cells at the apical surface and in the SVZ independently in images of 650 μm of width and calculated the percentage of PH3⁺ cells at the SVZ. 250μm × 250 μm images were taken in the neocortex for the PAX6-TBR2 co-staining. Each cell type was counted independently. To analyze the NeuN labeling at E14.5, the thickness of the NeuN+ layer was measured at least four times along the neocortex.

ADSCs differentiate into NSCs by neurosphere technique. In the first stage, ADSCs of the fourth passage were removed using trypsin and EDTA and plated with serum‐free DMEM/F12 containing 2% B27, 20 ng/mL of the epidermal growth factor (EGF), and 20 ng/mL of the basic fibroblast growth factor (bFGF) (Invitrogen, Paisley, Scotland). After 7 days, the cell aggregates (neurosphere) were harvested into single cells and the dissociated cells were then cultured in a T25 flask (2 × 10⁶ density) in a neurosphere medium with 10% FBS (NSC culture medium). NSCs were cultured on a six‐well plate and immunolabeled with primary antibodies against nestin (Abcam, ab11306), NF 68 (Abcam, ab223343), Neurod (Abcam, ab239955), Sox2 (Abcam, ab92494), Oct4 (Abcam, ab19857), and Neun (Abcam, ab177487) (Darvishi et al., 2020 (link); Moayeri et al., 2020 (link)).