Anti gfap

A separate group of animals underwent craniotomy and the TBI procedure and were infused with unlabeled 0.2 M β-hydroxybutyrate (BHB) (Sigma, Cat# 54965) (see surgery description as above). At 4 h post-TBI, animals were anesthetized with isoflurane and transcardially perfused with 0.9% saline (w/v), followed by the perfusion with fixative (4% paraformaldehyde in a phosphate buffer solution (PBS), pH 7.4). The brains were harvested, post-fixed overnight, and subsequently rehydrated with 30% sucrose in PBS. Free-floating 50 µm coronal brain sections were blocked for 1 h and 15 min in 20% natural goat serum, 1% bovine serum albumin, and 0.3% TX-100 in PBS. Sections were incubated with primary antibodies overnight at 4 °C; antibodies included Anti-BDH1 1:500 (Sigma; Cat# HPA030947, Lot R32980), Anti-NeuN 1:1,000 (Millipore, Burlington, MA, USA; Cat# MAB377, Lot 2140038), Anti-GFAP 1:750 (Abcam, Cambridge, MA, USA; Cat# ab4674, Lot GR1473-16). Secondary antibodies raised in goat (FITC anti-rabbit, TRITC anti-mouse, and Alexa Fluor 647 anti-chicken) were applied for 1.5 h at 1:200. Tissue was mounted using Prolong Gold with DAPI (Molecular Probes, Waltham, MA, USA; Cat# P36935). Images were obtained using a Zeiss LSM 510 Meta confocal microscope.

Mouse astrocytes cultured on cover glasses were fixed with 4% PFA, rinsed with PBS, and then blocked by 2% BSA in PBS. Cells were incubated overnight at 4 °C with primary antibodies anti-GFAP (1:1000; Abcam). Cover glasses were washed and incubated for 1 h at room temperature with secondary antibodies including anti-mouse IgG (coupled with Alexa Fluor 488, Life Technologies). Nuclear DNA was stained with DAPI. Cover glasses were mounted on glass slides with mounting buffer (Sigma-Aldrich). Morphological changes were visualized by a Zeiss 710 confocal laser scanning microscope.

About 10,000 cells/cm² (hADSC cultured in classical (collagenase + FBS) and cell therapy-ready (explant + hPL) conditions) were cultured in chamber slides (Ibidi, Baar, Switzerland) for 24 h before fixation with 4% paraformaldehyde at room temperature for 20 min. Samples were blocked in 2% BSA (Sigma Aldrich) in PBS and permeabilized with 0.1% Triton-X100 (AppliChem, Darmstadt, Germany) for cytosolic/nuclear antigens or with 0.1% Tween 20 (AppliChem) for surface antigens. After appropriate permeabilization, cells were incubated with the primary antibody anti-STRO-1 (1:400, Thermo Fischer Scientific, mouse monoclonal), anti-NES (1:200, Abcam, rabbit monoclonal), anti-GFAP (1:200, Abcam, rabbit monoclonal), and anti-MPZ (1:100, Abcam, rabbit polyclonal) at 4 °C. The following day, slides were incubated for 1 h at room temperature with FITC-conjugated secondary antibody (anti-mouse, 1:200, Abcam, polyclonal). Cell nuclei were labeled with Hoechst 33342 (Thermo Fischer, 20 mM) added to the secondary antibody solution at a concentration of 8 μM. Samples were examined under a fluorescence microscope at ×10 magnification (Olympus IX81, HamBurg, Germany). Experiments were conducted in technical and biological triplicates.

The AKT inhibitor LY294002, GSK3β inhibitor SB216763, NFκB inhibitor Bay 11–7082, FITC-dextran, and Evans blue were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, United States). JNK agonist anisomycin was purchased from Beyotime (Beyotime Biotechnology, Shanghai, China). TNF-α was obtained from R&D Systems (Minneapolis, MN, United States). The following primary antibodies were applied in this study: anti-p-GSK3β, anti-GSK3β, anti-p-JNK, anti-JNK, anti-p-NFκB and anti-NFκB antibodies purchased from Cell Signaling Technology (Danvers, MA, United States); anti-FGF20, anti-VE-cadherin, anti-Claudin-5, and anti-GFAP antibodies obtained from Abcam (Cambridge, MA, United States); and anti-p-AKT, anti-AKT and anti-Occludin antibodies purchased from Invitrogen (Carlsbad, CA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively. The secondary antibodies used in this study were goat anti-rabbit immunoglobulin G (IgG) H&L (HRP) and goat anti-mouse IgG-HRP purchased from Abcam (Cambridge, MA, United States) and Santa Cruz Biotechnology (Dallas, TX, United States), respectively.

Immunoblotting was performed as described previously^{24 (link)}. β-actin (Sigma, St. Louis, MO) was used as inter control. The antibodies used included anti-CYP24A1 (Abcam, UK), anti-GFAP (Abcam, UK), anti-Tuj1 (Sigma, USA), anti-Sox2 (Abcam, UK; Abclonal, Beijing, China), anti-CD133 (Miltenyi Biotec, Germany; Abclonal, Beijing, China), anti-Nestin (Abclonal, Beijing, China), anti-Oct4 (Abclonal, Beijing, China), anti-CNPase (Abclonal, Beijing, China), and anti-β-actin (Sigma, USA).

The mice were deeply anesthetized and transcardially perfused with pre-cooled PBS following 4% paraformaldehyde. The whole spinal cord was blown out using the hydraulic pressure method. The L₄–L₅ spinal cord was dissected and dehydrated in 30% sucrose for 2 days. The tissues were then frozen in O.C.T. and cut into 8 µm frozen sections using a cryostat (Leica Biosystems, Heidelberg, Germany). The sections were blocked with 0.3% Triton X-100 for 10 min and 5% goat serum for 1 h. They were then incubated with the primary antibodies overnight at 4 °C. The following primary antibody was used: anti-GFAP (1:200, Abcam). After rinsing three times with PBS, the sections were incubated with a fluorescence-labeled secondary antibody for 1 h. Images were collected using a fluorescence microscope (Olympus, Tokyo, Japan), and the analysis was performed using Image J software.