Anti mucin2

For histological analysis, 0.5 cm distal colon was fixed in Zinc Formalin (Polyscience Inc.) for 3 hours and then embedded in paraffin blocks. We then prepared 5 μm paraffin cross-sections, which were used for hematoxylin and eosin (H&E) staining (Mayer’s Hematoxylin solution, 1% Eosin Solution, Wako) following standard procedures. The histological images were captured using the BX51-P Polarizing microscope (Olympus) and processed with the Olympus D.P. Controller 2002 software.
For immunohistochemistry analysis, 0.5 cm distal colon was fixed overnight in 4% paraformaldehyde (PFA) (Wako) at 4°C and mounted in embedding medium Tissue-Tek O.C.T Compound (Sakura). The tissues were cut into 8 μm sections and permeabilized with 0.2% saponin (Nacalai Tesque) in PBS. The sections were then blocked with 5% goat serum (Wako) for Mucin 2 detection. Subsequently, the sections were stained with anti-Mucin2 (1:200, rabbit, clone: H-300, Santa Cruz Biotechnology) at 4°C overnight. For the second antibody, Alexa Fluor 488 conjugated donkey anti-rabbit IgG antibody (1:400, Thermo Fisher Scientific) was used with DAPI (1:1000, Dojindo). The sections were assessed using the Leica TCS SP8 (Leica Microsystems).

Immunofluorescence assay was executed as pronounced [61 (link)]. Tissue sections of 7 μm were incubated with the following primary antibody anti-Mucin 2 at 37°C overnight (1:100; Santa Cruz Biotechnology, Dallas, TX, USA). After the incubation with the primary antibody, the sections were washed with PBS and incubated with a secondary antibody Alexa Fluor 488 goat anti-mouse (1:1000 v/v Molecular Probes, UK) for 1 h at room temperature. 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt; Germany) 2 μg/ml in PBS was added for nuclear staining. The images were photographed at 40x magnification using an optical microscope (Zeiss, Axio Vision).

Human tissues were stained by fluorescence in situ hybridization using a 16S rRNA probe as previously described.^{42 (link)} Briefly, OCT-embedded tissue was cryo-sectioned at a 5 µm thickness and then fixed in 4% paraformaldehyde for 24 h at 4 °C. Formalin-fixed paraffin-embedded human polyp tissues were de-paraffinized with sequential washes in xylene and ethanol. Samples were then hybridized using the EUB338-cy3 probe (Eurofins Genomics) at 55 °C overnight. Immunofluorescence was performed with 5% BSA blocking, followed by staining with anti-Mucin-2 (Santa Cruz Biotechnology) and anti-E-cadherin (Cell Signaling Technology) at 4 °C overnight. Alexa Fluor 488 (Thermo Fisher) and Alexa Fluor 647 (Thermo Fisher) were used to detect Mucin-2 and E-cadherin, respectively. Nuclei were stained with 10 µg/mL DAPI (Sigma-Aldrich). Slides were mounted with Prolong Gold (Invitrogen). Images were taken at ×63/1.4 A magnification using a ZEISS LSM 880 Confocal Microscope maintained at the CCAM Microscopy Facility at the University of Connecticut Health Center. Images were captured with z-stack and 10% overlap tile scanning to obtain in-depth bacterial localization on each mucosal sample. Finally, images were processed with ImageJ to generate a final image.

ZO‐1 (Santa Cruz Biotechnology) was used at 1/100 for immunostaining; Phalloidin‐AF647 (Molecular Probes) was used at 1/200 for immunostaining; Laminin (Abcam) was used at 1/100 for immunostaining, mouse monoclonal against E‐cadherin (BD Transductions), and rabbit polyclonal anti‐Mucin‐2 (Santa Cruz Biotechnology) were used at 1:500 for immunofluorescence, and HAstv1 capsid antibody (Abcam) was used at 1/300 for immunostaining. Secondary antibodies were conjugated with AF488 (Molecular Probes), AF568 (Molecular Probes), and AF647 (Molecular Probes) directed against the animal source.

After medium removal, the organoids were fixed with 4% paraformaldehyde. Permeabilization was performed with 0.2% Triton X-100 followed by a blocking step using 5% BSA. The organoids were incubated with the following primary antibodies: anti-Lgr5 (Abgent, CA), anti-Ki67 (Cell Signaling Technology, MA), anti-Lysozyme (Diagnostic Biosystems, CA), anti-Mucin 2 (Santa Cruz Biotechnology, CA), anti-Chromogranin A (Santa Cruz Biotechnology), anti-Villin (Santa Cruz Biotechnology), and anti-cleaved caspase-3 (Cell Signaling Technology) at 4 °C overnight. Then, the organoids were incubated with the secondary antibodies, either Alexa Fluor 488-conjugated anti-mouse IgG (Life Technologies, MD) or Alexa Fluor 555-conjugated anti-rabbit IgG (Life Technologies), at room temperature for 2 h. The nuclei were stained with DAPI (Sigma-Aldrich) for 1 h, and the organoids were imaged using a confocal microscope (LSM 710; Carl Zeiss). The mean fluorescence intensity was analyzed with the ImageJ software, and the intensity of each marker was normalized to that of DAPI.