Sc 33725

Protein expressions in the ipsilateral hemisphere were measured by western blot as previously described [3 (link)]. Protein samples with equal volumes were loaded on an SDS-PAGE gel, and then electrophoresed protein bands were transferred to a nitrocellulose membrane. The membrane was incubated with the following primary antibodies overnight at 4 °C: rabbit polyclonal anti-frizzled 7 (1:1000; ab64636, Abcam, MA), rabbit polyclonal anti-WISP1 (1:2000; ab178547, Abcam, MA), mouse monoclonal anti-Dvl (1:200; sc-166303, Santa Cruz Biotechnology, TX), rabbit monoclonal anti-β-Catenin (1:10,000; ab32572, Abcam, MA), rabbit polyclonal anti-phospho-β-Catenin (Ser33/37/Thr41) (1:1000; #9561, Cell Signaling, MA), rat monoclonal anti-ZO-1 (1:200, sc-33725, Santa Cruz Biotechnology, TX), rabbit polyclonal anti-Claudin-5 (1:500; ab15106, Abcam, MA), and mouse monoclonal anti-VE-Cadherin (1:500; sc-9989, Santa Cruz Biotechnology, TX). Appropriate secondary antibody (Santa Cruz Biotechnology, TX) was applied to the membranes and incubated for 1 h at room temperature. Immunoblots were probed and exposed to films. Band densities were analyzed using Image J software (NIH, Bethesda, MD).

For hematoxylin and eosin (H&E) staining, paraformalin-fixed paraffin tissue sections were used to evaluate the characteristics of liver and intestinal tissues regarding histological changes and fibrosis. The immunohistochemistry procedure was performed as described previously [27 (link)], including the following primary antibodies CHOP, GRP78, PDI, and XBP-1 (sc-7351, sc-166490, sc-74551, and sc-8015, Santa Cruz Biotech, USA). The positive areas of CHOP, GRP78, PDI, and XBP-1 were recorded. For immunofluorescence, the tissue sections were blocked with 10% normal donkey serum for 1 h at 25° C in PBS and then incubated overnight with primary antibodies against claudin-1, occludin, and ZO-1 (sc-166338, sc-133256, sc-33725, Santa Cruz Biotech, USA) and P-gp (ab261736, Abcam) at 4° C. The nuclei were stained with Hoechst 33258 (0.25 l g/mL) dye. All fluorescence images were captured on a Nikon ECLIPSE Ti microscope.

Liver tissue extracts were prepared as described [58 (link)], and blots were probed with anti-TLR4 (1:1000) (SC-293072, Santa-Cruz Biotechnology, Dallas, TX, USA), anti-ZO-1 (1:1000) (SC-33725, Santa-Cruz Biotechnology), anti-Occludin (1:500) (SC-133256, Santa-Cruz Biotechnology), and anti-Actin (1:5000) (SC-1616, Santa-Cruz Biotechnology,) or anti-Gapdh (1:3000) (SC-47724, Santa-Cruz Biotechnology), as loading controls. Blots were developed with chemiluminescence reagent (Immobilon HRP-substrate reagent; Millipore, Burlington, MA, USA) and the corresponding images were acquired with the ChemiDoc-MP Imaging detection system (BioRad; CDMX, México). Images were further processed for densitometry analysis using ImageJ software (V 1.53c) [49 (link)].