Anti muc2

Intestinal tissue was collected and fixed overnight in 4% PFA in PBS and embedded in paraffin. In all, 5-μm sections were stained by HE and PAS for histological analysis and goblet cells staining. Sample were embedded in OCT and 8-μm sections were performed for immunofluorescence staining by using the following primary antibodies: TUNEL (Roche, 11684817910), anti-Ki67 (BD; 550609), anti-CD24 (Thermo Fisher; MA5-11828), anti-Muc2 (Santa Cruz; sc-515032), anti-CgA (Immunostar; 20086). All primary antibodies were used as 1:100 dilutions. Goat anti-mouse Alexa fluor®488 (Abcam, ab150113) and goat anti-mouse Alexa fluor®647 (ab150115) secondary antibodies were used at 1:500–1:1000 dilutions.
For fluorescence in situ hybridizations (FISH), tissues of jejunum were fixed overnight in 4% PFA, paraffin embedded, and sectioned at 10 μm. The probe sequences targeting Lgr5, Olfm4 and Lys (Lysome) was: Lgr5 probe (5′-FAM-GACGACAGGCGGTTGGACGATAGGT-FAM-3′), Olfm4 probe (5′-FAM-CACTGACACCTCGCCACCATTCCA-FAM-3′) and Lys probe (5′-CY3-GCACCGATCATAGACCTTGGCCTGTA-3′). The protocols used for in vitro transcription and in situ hybridization were previously described^{44 (link)}. All images were acquired and processed with Zeiss Axio-Imager Z1 with apotome or Leica SP5 confocal microscope.

Cells were seeded on 18 mm glass coverslips, fixed with 4% methanol-free paraformaldehyde, and blocked in 0.1% BSA + 0.01% TritonX-100 for 1 hr. Cells were probed with anti-CLCA1 (Abcam, ab180851, 1:100), anti-Muc2 (Santa Cruz, sc-515032, 1:200), anti-Rab7 (Sigma, R4779, 1:400), and Phalloidin-594 (Invitrogen, A12381, 1:400) at 4℃ overnight. Cells were further incubated with fluorophore tagged secondary antibodies for 2 hr. Nucleic acid was stained with DAPI (1 µg/ml). Coverslips were mounted with Prolong Gold Antifade Reagent (Thermo Fisher) and visualized in confocal microscope (Leica SP8) for immunocytochemistry and in Elyra PS1 (Carl Zeiss) for structured illumination microscopy.

Paraffin-embedded specimens were sectioned at a thickness of 4 μm and stained with hematoxylin and eosin (HE). For the histological assessment of intestinal development, the villus height and crypt depth of each intestinal section were measured under a light microscope (DM5000 B; Leica, Wetzlar, Germany), and 100 well-oriented, intact villus/crypt of each mouse were randomly selected for evaluation.
For immunohistochemistry, tissue microarray sections were deparaffinized with xylene, rehydrated with gradient concentrations of ethanol, and soaked in 3% hydrogen solution. Then, the sample was exposed to antigen hot retrieval with citrate buffer and further incubated with a blocking serum. After blocking, the tissue slices were incubated overnight with a monoclonal anti-Ki67 (ab16667; Abcam, Cambridge, UK), anti-Muc2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), or an anti-lysozyme (ab108508; Abcam) antibody. After incubation, the sections were washed with PBS, incubated with labeled polymer–horseradish peroxidase, and counterstained with 3,3′-diaminobenzidine. The sections were observed under a Leica microscope (DM5000 B; Wetzlar, Germany). Positive cells were counted by observing 100 villi or crypts per mouse from at least five randomly selected fields in a blinded manner. The original magnification of the HE and immunohistochemical staining was ×200.

Paraffin embedded sections (~5-μm thick) were deparaffinized by heating to 60 °C for 15 min, cleared with xylene, followed by an ethanol gradient (75%, 95%, 100%) and water and steamed for 30 min in citrate buffer for antigen retrieval. Tissues were then treated with blocking buffer (goat or donkey serum in PBS containing 1% bovine serum albumin [BSA], 0.1% Triton X-100, 0.05% Tween 20, and 0.05% sodium azide). The primary antibodies used were anti-Ki-67 (Thermo Scientific, Waltham, MA, USA), anti-Lysozyme (Santa Cruz, Dallas, TX, USA), anti-Muc2 (Santa Cruz), anti-CA-1 (Santa Cruz), anti-5HT (Antibodies Incorporated, Davis, CA, USA), anti-Claudin-3 (Invitrogen, Carlsbad, CA, USA), anti-Claudin-4 (Invitrogen). The secondary antibodies used were Alexa Fluor 568- or 488-conjugated goat anti-rabbit IgG, and Alexa Fluor 568- or 488-conjugated donkey anti-rabbit or donkey anti-goat IgG (Life Technologies, Carlsbad, CA, USA). ProLong gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA) to stain DNA was used to mount tissues. Tissues were viewed on a Zeiss Axio Imager microscope, and images were taken using AxioVision software and an AxioCam HRm camera.

Histopathological analysis was performed by using hematoxylin and eosin (H&E) staining. Immunohistochemical analysis of HER2 was performed to evaluate cell proliferation and downstream intracellular signaling in neoplastic tissue. Primary antibodies used in this study were anti-HER2 (1:200, CST#4290, Cell Signaling Technology, Danvers, MA, USA), anti-MUC1 (1:100, ab15481, Abcam, Cambridge, MA, USA), anti-MUC2 (1:200, sc-15334, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUC5 (1:100, sc-21701, Santa Cruz Biotechnology), anti-Ki67 (1:100, ab16667, Abcam), anti-CyclinD1 (1:25, CST#2978, Cell Signaling Technology), anti-phospho-p44/p42 (1:100, CST#4376, Cell Signaling Technology), anti-SOX-9 (1:100, sc-20095, Santa Cruz Biotechnology), anti-TP53 (1:200, CST#2524, Cell Signaling Technology), and anti-CK19 (1:50, sc-376126, Santa Cruz Biotechnology).