Monoclonal rabbit anti tgf β1

Cells were lysed with lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, and 1% (v/v) Triton™ X-100) containing protease and phosphatase inhibitor cocktails (Roche, Indianapolis, IN, USA). Total lysates were separated by SDS-PAGE using an 8% gel and then transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with the following primary antibodies: monoclonal mouse anti-Nanog (dilution 1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), polyclonal rabbit anti-Sox2 (dilution 1:1000; Merck Millipore), monoclonal rabbit anti-TGF-β1 (dilution 1:1000; Abcam, Cambridge, UK), monoclonal mouse anti-α-SMA (dilution 1:1000; Abcam), and monoclonal mouse anti-β-actin (dilution 1:10,000; Sigma-Aldrich). For lectin blot, the membranes were incubated with biotin-conjugated lectins: biotin-conjugated SNA, biotin-conjugated RCA120, biotin-conjugated LEL, and biotin-conjugated WGA. The membranes were then incubated with the appropriate peroxidase-conjugated secondary antibodies (dilution 1:30,000; Cell Signaling Technology), or peroxidase-conjugated streptavidin (Jackson ImmunoResearch Labs, West Grorve, PA, USA) for biotin-conjugated lectin, washed, and developed with ECL™ Prime reagents (GE Healthcare, Piscataway, NJ, USA).

Western blot analysis was performed as previously described [3 (link)]. White matter tissue adjacent to the hematoma was sampled. The primary and secondary antibodies were polyclonal rabbit anti-IL17A (Abcam, 1: 1000), polyclonal rabbit anti-IL21 (1: 500, Abcam), polyclonal rabbit anti-IL22RA2/IL-22BP (Abcam, 1: 1000), monoclonal rabbit anti-TGF-β1 (Abcam, 1: 1000), monoclonal rabbit anti-TNFα (Abcam, 1: 1000), polyclonal rabbit anti-IFNγ (Proteintech, 1: 500), monoclonal rabbit anti-Notch1 (Abcam, 1: 1000), polyclonal rabbit anti-ACT-1 (Proteintech, 1: 1000), monoclonal rabbit anti-RBP-J (Abcam, 1: 1000), and monoclonal rabbit anti-NICD1 (CST, 1: 1000).

Sample collection and western blotting were performed as previously reported^{36 (link),47 (link)} with the following primary antibodies: anti-VEGFA antibody (Abcam), mouse monoclonal anti-α-SMA (Sigma-Aldrich), rabbit monoclonal anti-TGF-β1 (Abcam), IDO1 polyclonal antibody (Proteintech, Rosemont, IL, USA), mouse monoclonal anti-Foxp3 (Abcam), rabbit polyclonal anti-CD4 (Abcam), and mouse monoclonal anti-GAPDH (Sigma-Aldrich). Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako) or goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura or Pico Systems (Thermo Fisher Scientific, Waltham, MA, USA) were used to detect signals. The intensity of each band was analyzed by ImageJ software and standardized to the level of GAPDH.