Arg22476

The ileum was taken from 10 cm of the intestine in front of the ileocecal valve and about 4 cm of ileum was isolated, fixed in 4% paraformaldehyde and embedded in paraffin to dissected into 5-μm-thick slides. Subsequently, hematoxylin and eosin (H&E) were performed on the sections of ileum for the analysis of inflammatory injury. Six random fields were selected from each section, and the histopathological evaluation of inflammatory and crypt damage was assessed as previously described. The ileum sections were also stained against F4/80 (ARG22476, Arigo Biolaboratories Corp, Taiwan, China) and CD11c (ARG59698, Arigo Biolaboratories Corp, Taiwan, China) for the immunofluorescence analysis and against NF-κB p65 (phosphor S536) (ab86299, Abcam, Cambridge, United Kingdom) for the immunohistochemistry analysis. Images were captured with a Mirax scanner (3DHISTECH, Budapest, Hungary), and the fluorescence intensities and positive area were analyzed with Image J software.

About 4 cm of ileum was fixed in 4% paraformaldehyde to prepare 5 μm paraffin slides. The ileum sections from the 24 mice at the end of experiment were stained with hematoxylin and eosin (H&E) for the analysis of inflammatory changes (n = 8). Histopathological assessment of inflammatory and crypt damages was assessed as previously stated by a light microscope (Dieleman et al., 1998 (link)). Six randomly selected fields from each slides were analyzed. The ileum sections were also stained with the first antibodies against F4/80 (ARG22476) and CD11c (n = 5) (ARG59698; Arigo Biolaboratories Corp, Taiwan). For immunohistochemistry analysis, we used Anti-NF-κB p65 (phospho S536) (ab86299, Abcam, Cambridge, United Kingdom) (n = 5). Images were captured with a Mirax scanner (3DHISTECH, Hungary), and the area of positive points was calculated with Image Pro (MediaCybernetics, Rockville, MD).

Paraffin sections were dewaxed in xylene and rehydrated in ethanol, respectively. Antigen retrieval was performed in a microwave oven for 2 min at high power and then for 10 min at low power, followed by cooling down to room temperature. The activity of endogenous peroxidase was quenched with 3% H₂O₂ for 10 min. Tissue blocking was performed with goat serum for 1 h at room temperature. Tissue sections were then incubated at 4°C over-night with primary antibodies for T lymphocytes (CD3, ARG52744, Arigo), helper T lymphocytes (CD4, ab183685, Abcam), cytotoxic T lymphocytes (CD8, ab217344, Abcam), Treg lymphocytes (FOXP3, GB11093, Servicebio), natural killer cell (CD49b, AGR57601), or macrophages (F4/80, ARG22476, Arigo). The sample were then incubated with horseradish peroxidase labeled secondary antibody (1: 500) for 1 h at 37°C. Color development was performed using the DAB peroxidase substrate kit (ZSGB-BIO) according to the manufacturer’s instruction. The sections were finally dehydrated in ethanol and xylene, and sealed with neutral resin. The image signal recorded by microscope was converted into digital signal by Image J to analyze the distribution and staining intensity of positive markers.