Sc 166338

Mice were perfused with 20 mL of cold 0.1 M PBS and subsequently with 4% PFA as described earlier while under deep anesthesia. The brains were removed, fixed with PFA overnight, and treated with 30% sucrose at 4°C for an additional 72 h. Ten-micrometer coronal sections were then cut for further analysis. The mouse colons were fixed with 4% PFA overnight. Sections (5 μm) were cut, washed in PBS, and blocked with 10% donkey serum with 0.3% Triton X-100 (room temperature, 1 h) before incubation with anti-Iba-1 (1:500, Abcam, ab5076), anti-CD16 (1:100, Abcam, ab25235), anti-Arg-1 (1:500, Proteintech, 16001-1-AP), and anti-claudin (1:50, Santa Cruz Biotechnology, sc-166338) antibodies (4°C, overnight). After rewashing, the sections were incubated with species-appropriate anti-IgG antibodies conjugated with Alexa Fluor 488, 555, or 594 at 37°C for 1 h and stained with DAPI (Abcam, ab104135). Three sections per animal were assessed under fluorescence microscopy (Leica, Mannheim, Germany). Target cells in brain sections were observed in three fields, the means were determined, and the fluorescence intensities in the colonic sections were analyzed by ImageJ software (NIH).

The colon was collected from rats and flushed of fecal content with ice-cold phosphate-buffered saline (PBS), as described previously [60 (link)]. Tissues were minced and homogenized using a Potter-Elvehjem Grinder homogenizer on ice in 20% (w/v) TNE lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 1% NP-40, 1% SDS, 0.1% DOC) with protease and phosphatase inhibitors. Samples were then sonicated and boiled for 5 min at 95 °C. Proteins were quantified with the Bradford assay. Total lysates were run on a 4–20% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA) and then transferred to PVDF membranes (Trans-Blot TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against claudin-1 (sc-166338, Santa Cruz, Dallas, TX, USA), occludin (ab167161, Abcam, Cambridge, MA, USA), and ZO-1 (ab96587, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721) phospho-AMPKα (BK2535T, Cell Signaling), AMPKα (BK5831T, Cell Signaling). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by IBright Analysis software (version, 1.2.2, Waltham, MA, USA).

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.

The Claudin-1 protein expression was determined in the ascending and descending colon tissue. The resected tissue was fixed immediately with 10% formaldehyde in 1X phosphate buffer saline (PBS), processed, and embedded in paraffin. The paraffin-embedded samples were cut and the sections (3-µm) were treated with the Novolink^® polymer detection system (Leica Biosystems, Buffalo Grove, IL, USA), according to the manufacturer’s instructions. The antigen retrieval was performed with an EDTA buffer (1 mM EDTA, 0.05% Tween 20, and pH 9.0) for 40 min.
The sections were incubated overnight at 4 °C with a mouse monoclonal primary antibody (sc-166338; Santa Cruz Biotechnology, Danvers, MA, USA) at a 1:250 dilution. The adipose tissue was used as the negative control. Finally, the chromogen working solution and hematoxylin were employed to perform the detection and the counterstain, respectively. The Claudin-1 expression was documented using Aperio LV1 IVD equipment and Aperio image scope software (Leica Biosystems, Buffalo Grove, IL, USA). The staining intensity was scored as follows: 0 = negative; 1 = weak; 2 = moderate, and 3 = strong.