Gb113373

In order to evaluate the infiltration of inflammatory cells in pancreas, we detected CD45, Ly6G and F4/80 by immunofluorescence. Briefly, Frozen slides are baked in a 37 °C oven for 10–20 min and fix in paraformaldehyde for 30 min. Afterward, the slides were heated in an autoclave within EDTA antigen retrieval buffer (pH 8.0) for antigen repairing, followed by 3% BSA to block non-specific binding. Slides were then incubated with primary antibody including CD45 (GB11066, Servicebio, China, 1:1000), Ly6G (GB11229, Servicebio, China, 1:500) and F4/80 (GB113373, Servicebio, China, 1:500) at 4 °C overnight, and with Cy3 conjugated secondary antibody (GB21303, Servicebio, China, 1:500) at room temperature for 50 min in dark condition. After DAPI counterstain in nucleus, the sections were observed and images collected under the fluorescence microscope. The mean fluorescence intensity (MFI) was analyzed by ImageJ (V1.8.0.112). Five visual fields were randomly selected on each slide for MFI analysis.

Photosensitive resin was purchased from Ausbond (Shenzhen, China). Hair removing cream of VEET was obtained from Reckitt Benckiser Plc. (Wuhan, China). Isoflurane was purchased from Shenzhen Reward Life Technology Co., Ltd. (Shenzhen, China). MXD liniment (2%) was purchased from Sichuan Medco Huakang Pharmaceutical Co., LTD. (Sichuan, China). DAPI-containing antifluorescence quenching tablets were obtained from Beyotime Biotechnology (Shanghai, China). Cell-Counting Kit-8 was purchased from Dalian Meilunbio Co. Limited. (Dalian, China). Clodronate disodium was obtained from ShangHai YuanYe Biotechnology Co., Ltd. (Shanghai, China).
The following antibodies were used for immunostaining: primary antibody: anti-β-Catenin (Wanlei bio, WL 0962a, 1:200), anti-CK15 (HuaBio, ET1609-54, 1:100), anti-Ki67 (ThermoFisher Scientific, 14-5698-80, 1:200), and anti-F4/80 (Servicebio, GB113373, 1:500). Secondary antibodies: fluorescence conjugates with rhodamine (ZSGB-BIO, 1:100), Cy3 (Servicebio, 1: 200), and FITC (ZSGB-BIO, 1:100).

First, the skull samples were decalcified by immersing them in a test tube, containing the ethylene diamine tetraacetic acid solution (pH 7.2, Biosharp, China). The test tube was then placed at 37°C in a shaking incubator. The decalcifying solution was refreshed every 2 days to speed up the decalcification process. The skull specimens were paraffinized after decalcification and sectioned. After dewaxing the paraffin sections, the immunofluorescence staining was performed as described in the literature [34 (link)]. The following antibodies were used in this study: F4/80 antibody (macrophage marker, GB113373, 1:300, Servicebio, China), iNOS antibody (M1-type macrophage marker, GB11119, 1:300, Servicebio, China), CD206 antibody (M2-type macrophage marker, GB13438, 1:300, Servicebio, China), TNFα antibody (GB23303, 1:2000, Servicebio, China) and IL10 antibody (GB25303, 1:200, Servicebio, China). After staining, a fluorescence microscope (Nikon, Japan) was used to observe and photograph the sections. The average fluorescence intensity of each sample in the images was analyzed using the ImageJ software.

Mice were transcardially well-perfused with 0.9% saline infusion followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The tissues of the colons and the brains were dissected and immediately placed in 4% paraformaldehyde for 24 h, and next in 4% paraformaldehyde containing 30% sucrose. The brain and the colon tissues were embedded in paraffin and sliced into 3-μm-thick and 5-μm-thick slides, respectively. Briefly, the slides went through antigen retrieval using sodium citrate solution (pH 6.0). After being blocked with 3% bovine serum albumin (Servicebio, G5001), the slides were incubated with primary antibodies at 4 °C overnight. The primary antibodies include the following: anti-F4/80 (Servicebio, GB113373, 1:5000 dilution), anti-ZO-1 (Servicebio, GB111981, 1:200 dilution), anti-IL-23 (Wanleibio, WL01655, 1:200 dilution), and anti-Iba-1 (Abcam, Ab178847, 1:500 dilution). Then, the appropriate secondary antibody goat anti-rabbit IgG-Cy3 (Servicebio, GB21303, 1:300 dilution) was used to detect the corresponding primary antibodies. DAPI solution (Servicebio, G1012) was used to detect nuclei. The representative images were captured by a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan). The quantification of positively stained cells was conducted using ImageJ.

For immunofluorescence staining, lung tissues were fixed with 4% paraformaldehyde overnight at 4°C and subsequently embedded in optimal cutting temperature compound (OCT). Nonspecific antigens were blocked with serum for 30 minutes at room temperature. To identify M1 and M2 macrophages in the lung tissues, anti-CD86 (GB13585, Servicebio) and anti-CD163 (GB112634, Servicebio) antibodies were used at a dilution of 1:50. Additionally, F4/80 (GB113373, Servicebio) was employed as a pan-macrophage marker at a concentration of 0.5 μg/mL. Nuclei were counterstained with DAPI (ab104139, Abcam). The obtained images were captured and analyzed using an inverted confocal laser scanning microscope (LSM 710, Carl Zeiss, Germany). Quantitative analysis was performed using ImageJ software.