Ab76774

The paraformaldehyde-fixed gastric tissues were embedded in paraffin and cut into sections, dewaxed and hydrated. After blocking the appropriate antisera, the sections were incubated with CDX2 (1:100 dilution; Abcam, ab157524), MUC2 (1:100 dilution; Abcam, ab76774), ki-67 (1:100 dilution; Abcam, ab16667), p53 (1:100 dilution; Millipore, #CBL404), and PTEN (1:100 dilution; Abcam, ab238032). Then, the sections were incubated with anti-rabbit or anti-mouse IgG (Boshide Biological Technology, Wuhan, China). Positive signals were visualized using a diaminobenzidine kit (GTVisionTM III Detection System/Mo&Rb; Gene Group Holdings, Shanghai, China), and the sections were counterstained with haematoxylin. Image-Pro was used to analyse the integrated optical density (IOD) and area of each picture. The mean density (IOD/area) was used to analyse the protein expression.

Colon tissues from experimental mice or cultured ex vivo colonic explants from normal mice were homogenized in ice-cold RIPA buffer containing protease inhibitors. The cleared lysates were obtained by centrifugation at 10,000 rpm at 4 °C for 15 min. Protein quantification was carried out using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, USA) with bovine serum albumin as a standard. Equivalent amounts of protein from each sample were separated by 10% SDS–PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Subsequently, the membranes were blocked in 5% milk, probed overnight at 4 °C with primary antibodies and then incubated with HRP-conjugated secondary antibodies. The primary antibodies included rabbit anti-Muc2 (ab76774, Abcam), rabbit anti-occludin (ab168986, Abcam), rabbit anti-ZO-1 (H-300, Santa Cruz), rabbit anti-NLRP6 (PA5-21022, Thermo Fisher Scientific), rabbit anti-caspase-1 (sc-514, Santa Cruz), rabbit anti-NLRP3 (#15101, CST), rabbit anti-PPAR-γ (#2435, CST), and mouse anti-β-actin (#4970, CST). The signals were detected with an enhanced chemiluminescence system (Tanon, Shanghai, China). The immunoreactive bands were quantified via densitometry using ImageJ (Version 1.50b, National Institutes of Health, USA) and standardized to β-actin and were expressed as fold changes relative to the control value.

ELISAs to measure the level of Mucin 5 ac (Muc5ac) and Muc2 in the airway and colon lavage fluid were adapted from previously published protocols (37 (link), 38 (link)). Briefly, plates were pre-incubated with carbonate buffer (pH 9.5). Airway and colonic lavage samples were then added to the carbonate buffer and incubated at 37°C overnight. Plates were blocked with 1% BSA PBS. Mucin was detected with anti-Muc5ac antibody (45M1, Thermo Fisher Scientific, UK) or anti-Muc2 antibody (ab76774, Abcam, UK). For Muc5ac, goat α-mouse HRP secondary antibody was used; for Muc2, goat α-rabbit HRP secondary antibody was used. ELISAs were developed using TMB (Thermo Fisher Scientific, UK) and stopped using 2 N H₂SO₄. Plates were read at 540 nm using a Fluostar (Omega).

To perform immunofluorescence staining on the colon tissue sections, the frozen sections embedded in OCT were fixed in 8% neutral buffered formalin for 30 min. After fixation, the sections were washed three times with distilled water (ddH2O) to remove residual formalin. For the staining process, primary antibodies specific to the target proteins of interest, such as anti-mouse Muc2 (ab76774, Abcam), CRAMP (sc-66843, Santa Cruz), or β-Defensin 2 (ab203077, Abcam), Ki67 (ab16667, Abcam), IL-1β (AF-401-NA, R&D) antibodies and anti E. coli antibody (ab25823, Abcam), were applied to the sections. Following the primary antibody incubation, secondary antibodies conjugated with biotin (ab6720, ab208000, ab207997, ab207996, Abcam and BAF109, R&D), were applied to the sections followed with streptavidin-FITC (405202, BioLegend) or PE (405204, BioLegend), allowing for the visualization of the target proteins. The 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei. The fluorescence intensity of all immunofluorescence staining was measured by ImageJ.

Formalin-fixed, paraffin-embedded 4 μm sections of lungs and intestinal tissue from H1N1-infected and DSS-treated mice were used for IHC. After deparaffinization with xylene, three sections from each group were incubated in a citrate buffer solution for antigen retrieval (20 min). Subsequently, the tissues were blocked with 5% bovine serum albumin for 30 min and then incubated at 4 °C for 24 h with 50 μL of primary antibodies against zonula occludens protein 1 (ZO-1, ab96587, abcam, Cambridge, UK); Mucin-2 (MUC2, ab76774, abcam, Cambridge, UK); Ki67 (ab15580, abcam, Cambridge, UK); toll-like receptor 4 (TLR4, ab22048, abcam, Cambridge, UK); and NOD-like receptor thermal protein domain associated protein 3 (NLRP3, ab214185, abcam, Cambridge, UK) at a 1:100 dilution. The following day, sections were incubated with the secondary antibody, goat anti-rabbit IgG (A0208, Beyotime, Shanghai, China), at 1:300 for 1 h at room temperature 22 °C. Finally, the samples were sealed with neutral balsam, and images were acquired using an Olympus SLIDEVIEW VS200 microscope (Olympus Corporation, Tokyo, Japan).