Anti claudin 1 antibody

Recombinant murine IL-22 was purchased from Peprotech (Cranbury, NJ, USA). Purified anti-mouse CD3ε (145-2C11) and its isotype control Armenian hamster IgG for the injection were obtained from BioLegend (San Diego, CA, USA) and BioXCell (Lebanon, NH, USA), respectively. Purified monoclonal anti-mouse IL-22 (IL22JOP) and its isotype control rat IgG2a used for in vivo administration were purchased from eBioscience and BioXCell, respectively. For immunohistochemistry, rabbit polyclonal anti-claudin-1 antibody was purchased from AbCam (Waltham, MA, USA). For flow cytometry, fluorescence conjugated antibodies specific for CD45 (clone 30-F11), CD4 (GK1.5), CD8α (536-7), CD19 (1D3), TCR-β (H57-597), IL-22 (Poly5164) and CD16/32 (clone 93) antibodies were purchased from BioLegend.

FFPE blocks of human colon specimens were obtained from the Department of Pathology at the University of Michigan (IRB HUM00038437). Colonoids and mouse colon tissue sections were prepared as described above. Sections were deparaffinized, and antigen retrieval was performed by boiling slides in a sodium citrate buffer. Blocking was performed by incubating sections with 10% normal serum for 1 h at RT. Sections were first stained by incubating with 5 μM of RTS*-Cy5.5 for 5 min followed by washing 3× with PBS. Next, antibody staining was performed by incubating with anti-claudin-1 antibody (Abcam, #ab15098) at 4 °C. Goat anti-rabbit IgG antibody conjugated with AF488 (Life Technologies (Carlsbad, CA ,USA), #A-11029) was used as a secondary antibody and incubated with the tissue sections for 2 h at RT. For human cytokeratin staining, sections were incubated with anti-cytokeratin (CAM 5.2, BD Biosciences, Franklin Lakes, NJ, USA, #345779) at RT for 1 h followed by incubation with goat anti-mouse IgG conjugated with AF488 (Thermo Fisher Scientific, #28175) for 2 h at RT. Fluorescence images were collected using an inverted confocal microscope (Leica (Deerfield, IL, USA), #SP5). The average fluorescence intensity was measured using custom Matlab R2022a (Mathworks) software.

Lipopolysaccharide was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). IL-1β, IL-6, TNFα, MCP-1, and ICAM-1 ELISA kits were purchased from Genetimes Technology Inc. (Shanghai, China). Anti-TH antibody, anti-PSD-95 antibody, anti-COX-2 antibody, anti-Claudin-1 antibody, anti-Occludin antibody, and anti-CX43 antibody, anti-HMGB1 antibody, anti-TLR4 antibody, and Cy3-conjugated secondary antibody were the products of Abcam (Cambridge, United Kingdom). Anti-Iba-1 was the product of Wako (Osaka, Japan).

Colonoid and tissue sections were processed for IHC staining. Deparaffinization and antigen retrieval were performed on unstained sections prior to staining as described previously [7 (link)]. Sections were immersed in 3% H₂O₂ for 30 min, blocked with normal serum for 30 min, and incubated overnight with anti-claudin-1 antibody (Abcam, Boston, MA, USA, #ab15098) at 4 °C. Antigen binding was detected with a VECTASTAIN Elite ABC HRP Kit containing biotinylated rabbit IgG (Vectorlabs (Mowry Ave Newark, CA, USA), #PK6101) and chromogen 3,3′-diaminobenzidine (DAB; Sigma #D3939) per the manufacturer’s instructions. Slides were counterstained with Harris hematoxylin. The digital images were collected using a standard brightfield/epifluorescence microscope (Zeiss (New York, NY, USA), #Axioskop 2 plus).

Common bile duct specimens were weighed, and 100-mg samples were placed in 1 mL RIPA extraction buffer and 10 μL phenylmethanesulfonyl fluoride (PMSF), ground up, and centrifuged at 17,226 g for 30 min. The supernatant was collected and stored at −20 °C until use. Protein concentration was detected using the BCA method (Pierce, USA). SDS-PAGE was performed using a 5 % stacking gel and 15 % separation gel. Target protein bands were transferred to a nitrocellulose membrane, blocked with non-specific antibody, incubated overnight at 4 °C with anti-occludin antibody (1:250; Abcam), anti-claudin-1 antibody (1:200; Abcam), and anti-MLCK antibody (1:400; Abcam). After incubation with HRP-conjugated secondary antibody, the protein bands were visualized with enhanced chemiluminescence. Band intensities were quantified using digital imaging analysis software (Tanon-1600, China).