Gtx100664

Paraffin sections were incubated overnight with primary antibodies in 1% horse serum albumin (PK-6200, Vector) at 4°C. The next day, the samples were washed with PBS and incubated with secondary antibodies at 25°C for 1 h. After washing with PBS, the sections were developed using ImmPACT^® NovaRED^® Substrate, Peroxidase (HRP) (SK-4805, Vector) for color reaction, and then observed under a microscope. The number of positive cells in 30 complete crypts was counted and expressed as the mean ± SD. Three mice were used in each group. The antibodies used were anti-BrdU (5292, CST), anti-Ki67 (9129, CST), anti-Cyclin D1 (2978, CST), anti-Lgr5/GPR49 (MAB8240, R&D Systems), anti-Sox9 (82630, CST), anti-Lysozyme (ab108508, Abcam), anti-Chromogranin A (GTX113165, GeneTex), and anti-Muc2 (GTX100664, GeneTex).

The cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100 for 10 min. After being blocked with 10% normal donkey serum, the cells were incubated with a primary antibody against MUC2 (1:1,000, GTX100664, Genetex, Irvine, CA, USA) at 4 °C overnight. Goat anti-rabbit IgG (H + L), DyLight 488 (1:1,000, 35552, Thermo Scientific) was used as a secondary antibody, and DAPI (1:10,000) was applied to counterstain the nuclei for 10 min at room temperature. Samples were washed with PBS three times for 5 min, and the coverslips were placed onto slides and mounted using VECTASHIELD® (Vector Laboratories, Burlingame, CA, USA). Images were obtained using a Nikon C1 plus confocal laser scanning microscope (Nikon, Tokyo, Japan).

The cells were fixed with 4% paraformaldehyde at 4 °C overnight. A permeabilization step with 0.1% Triton X-100 was performed for 10 min followed by blocking with 10% normal donkey serum (GTX73205, Genetex, Irvine, CA, USA). The cells were incubated with a primary antibody against MUC2 (1:1000 dilution; GTX100664, Genetex) at 4 °C overnight. Goat anti-rabbit immunoglobulin G (IgG) (H + L), DyLight 488 (1:1000 dilution; 35552, Thermo Scientific, Wilmington, DE, USA), was used as the secondary antibody for green fluorescence, and DAPI (1:10,000 dilution) was used to counterstain the nuclei for 5 min at room temperature. After washing samples with phosphate-buffered saline (PBS) three times for 5 min each, the slides were mounted using VECTASHIELD^® (Vector Laboratories, Burlingame, CA, USA). Images were captured using a Nikon C1 plus confocal laser scanning microscope (Nikon, Tokyo, Japan), and the intensity of green fluorescence indicating MUC2 protein was quantified by Olympus fluoview FV1000 ver.2.1b (Olympus, Tokyo, Japan) followed by normalizing the detected area and expressed as (%) compared to the negative control (100%).