Halo 101 wl halo 1

Mouse skin lesion tissues were fixed with 4% paraformaldehyde and embedded in paraffin, and Sect. (5 μm) were prepared, microwave oven antigen repair in citrate buffer, endogenous peroxidase was blocked by 3% hydrogen peroxide, serum was blocked. The primary antibody was incubated with MCP-1 (1:100, cat no: ab30852, Abcam, UK) and MIF (1:200, cat no: bs-1044R, Bioss, Beijing) at 4℃ overnight, and then incubated with secondary antibody (1:100, cat no: GB23303, Servicebio, Wuhan) at 37℃. DAB was added for color development, and hematoxylin was added for 3 min, and the slices were sealed with neutral gum. Images of the sections were acquired using a microcamera system (BA400Digital, Motic, Shanghai), and the % DAB Positive Tissue per image was calculated using the Halo data analysis system (Halo 101-WL-HALO-1, Indica labs, USA).

All sections embedded in paraffin were treated with xylene. Following dewaxing, the sections underwent dehydration using a graded series of ethanol solutions. After washing the sections with PBS for 5 minutes, they were incubated for 20 minutes with nonimmune goat serum (SP9001, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China). Subsequently, the sections were probed with mouse anti-NeuN polyclonal antibody (#94403, 1:100, Cell Singnaling Technology, Massachusetts, USA), anti-IL-1β (#BS-0812R, 1:100, Beijing Bioss Biotechnology Co, Ltd, China), and anti-IL-18 (#DF6252, 1:100, Affinity Biosciences, China), overnight at 4℃, and then with the secondary antibodies CY3-labeled goat anti-mouse IgG (#GB21301, 1:100, Servicebio) and FITC-labeled goat anti-rabbit IgG (#GB22303, 1:100, Servicebio) at room temperature for 30 minutes. The nucleus was labeled with DAPI. Finally, the microscopic camera system (OlyVIA, OLYMPUS, Japan) and halo data analysis system (Halo 101-WL-HALO-1, Indica labs, USA) were used for image acquisition and counting.