Gb111981

The paraffin-embedded tissues were sectioned into 3-μm-thick slides. According to standard histological protocols, hematoxylin and eosin (HE) staining was conducted. For immunohistochemistry (IHC), the following primary antibodies were used to incubate the slides, respectively, after blocking: bacterial lipopolysaccharide (LPS; HycultBiotech #HM6011, Netherlands), lipoteichoic acid (LTA; Santa Cruz, #sc-57752, United States), occludin (Servicebio #GB111401, China), and zonula occludens 1 (ZO-1; Servicebio #GB111981, China). IHC quantification involved the calculation of positive areas (occludin and ZO-1) using Aipathwell software (Version 2.0, Servicebio, China).

We procured colon tissue samples and prepared them for paraffin embedding. The tissues underwent a standard protocol where they were dewaxed using xylene, followed by dehydration through a series of graded ethanol solutions. Subsequently, a 0.01 mol/L citric acid buffer was applied for microwave-assisted antigen retrieval. Once the colon tissue sections were rinsed with PBS, we applied goat serum (16210064, Gibco, Grand Island, NE, USA) to block non-specific binding, allowing it to act for 10 min at ambient temperature. Subsequently, we introduced primary antibodies, specifically, Occludin (1:100, ab216327, Abcam, Cambridge, UK) and ZO-1 (1:100, GB111981, Servicebio, Wuhan, China), and left them to bind overnight in a 4 °C environment. For detection, we employed suitable secondary antibodies (A48255, Alexa Fluor 488 or 594, Invitrogen, Carlsbad, CA, USA), and after that, a 5-min room temperature incubation was carried out using 1 mg/mL DAPI (D1306, Invitrogen, Carlsbad, CA, USA) to visualize the nuclei. Visualization and imaging of these sections were performed using a Nikon Inverted fluorescence microscope (Model TE-2000U; Nikon, Tokyo, Japan), while the images were captured via SPOT digital camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA).