Ab15098

The nerve conduits were harvested and fixed in 4% PFA at 4 °C for 2 h. After being washed with PBS, the tissues were cryoprotected with 30% sucrose in PBS at 4 °C overnight, and were then embedded in optimum cutting temperature compound, followed by being frozen at − 80 °C. The frozen samples were cryosectioned longitudinally and transversely at − 20 °C for the thickness of 10 μm. The slices were placed onto Superfrost plus slides and stored at − 20 °C. IHC staining was performed for histological analysis. The slices were permeabilized with 0.5% Triton X-100 in PBS for 30 min, blocked with 4% normal goat serum in PBS for 1 h, and then incubated overnight at 4 °C with primary antibodies. The slides were then washed with PBS and incubated with secondary antibodies for 1 h at room temperature. After further PBS washing, the coverslips were mounted and viewed with a fluorescent microscope (Zeiss). The primary antibodies used for IHC analysis in this study were as follows: neural filament-medium polypeptide (NFM) (ab7794, Abcam), S100β (ab4066, Abcam), Claudin-1 (ab15098, Abcam), CD31 (ab28364, Abcam), ⍺-SMA (ab5694, Abcam), and CNN1 (ab233854, Abcam). Fluorescence-tagged anti-mouse and anti-rabbit secondary antibodies (Abcam) were used.

Western blotting of ileum and colon were performed as previously described^{61 (link)}. The primary antibodies against claudin 1 (1:1000 dilution, ab15098), occludin (1:1000 dilution, ab31721), and ZO-1 (1:1000 dilution, abG041) were purchased from Abcam.

The following primary antibodies were employed (dilution): ZO1 (1:1000, ab96587, Abcam, USA), occludin (1:1000, ab167161, Abcam, USA), claudin-1 (1:1000, ab15098, Abcam, USA), IκBα (1:2000, 4812, Cell Signaling Technology, USA), p-IκBα (1:2000, 2859, Cell Signaling Technology, USA), NF-κB p65 (1:1000, ab16502, Abcam, USA), monocyte chemotactic protein-1 (MCP-1, 1:1000, ab7202; Abcam, USA), cyclooxygenase-2 (COX-2, 1:1000, ab62331; Abcam, USA), Keap1 (1:1000, ab139729, Abcam, USA), Nrf2 (1:1000, ab31163, Abcam, USA), heme oxygenase 1 (HO-1, 1:2000, ab68477, Abcam, USA), catalase (1:1000, ab52477, Abcam, USA), NAD(P)H quinone dehydrogenase 1 (NQO1, 1:1000, ab28947, Abcam, USA), α smooth muscle actin (α-SMA, 1:300, ab7817, Abcam, USA), collagen I (1:5000, ab34710, Abcam, USA), and fibronectin (1:1000, ab2413, Abcam, USA). Western blot analysis was performed as previously described^{24 (link),26 (link),27 (link)}. Blots were obtained with ECL reagent and protein concentrations were normalized by actin expression. Specific bands indicating target proteins were analyzed using ImageJ 1.48 v software.