Fluorescein isothiocyanate (fitc)

Immunofluorescence (IF) analysis was performed using paraffin-embedded sections. Paraffin sections were dehydrated in sequential order. Auto-fluorescence was quenched with BSA. The samples were incubated with primary antibodies and secondary antibodies (Alexa 488 and FITC, Servicebio, Wuhan, China) at a dilution of 1:5000, and then mounted by DAPI staining reagent (Servicebio). Samples were visualized under a Pannoramic Scanner system (Pannoramic DESK, P-MIDI, P250, 3D HISTECH, Hungary).

After behavioral tests, mice were perfused. They were sequentially perfused with 40 ml of normal saline and 40 ml of a 4% paraformaldehyde solution (0.1 mol/L PBS, pH 7.4) through the ascending aorta after anesthesia with chloral hydrate. The mouse brain was fixed in 4% paraformaldehyde solution for more than 24 h, then dehydrated in gradient alcohol. Brain slices (thickness: 4 µm) were cut using a Frozen platform (JB-L5, Wuhan Junjie Electronics Co., Ltd., China) and collected in an EDTA antigen retrieval solution (pH 8.0). Next, the brain slices were rinsed with PBS (pH 7.4) three times for 5 min, then placed in 3% BSA and incubated at room temperature for 30 min. The brain slices were transferred into PBST with a primary antibody (Anti-P2X7R, 1:200, Bioss; Anti-Iba1, 1:500, Servicebio; Anti-Brdu, 1:200; Servicebio) and incubated at 4°C overnight. After rinsing with PBS (pH 7.4) for 5 min three times, the brain slices were transferred into PBS (pH 7.4) with a secondary antibody (FITC, 1:400, Servicebio; Cy3, 1:400, Servicebio) and incubated in the dark at room temperature for 50 min. For immunofluorescence staining, the patches were rinsed three times with PBS for 5 min three times, then dried with a 4’, 6-diamidino-2-phenylindole staining solution. Images were obtained using an Ortho-Fluorescent Microscopy (Nikon Eclipse C1, Nikon, Japan).

After the HaCaT cells were seeded on confocal dishes and the density approached 60%, the related modeling and treatments were conducted as described previously. The samples harvested were fixed by 4% paraformaldehyde for 30 min, 0.5% Triton X‐100 for 10 min, and 5% sheep serum for 1 h. Diluted primary antibodies of different concentrations were incubated at 4 °C overnight (α‐smooth muscle actin (α‐SMA), 1:500; fibronectin, 1:300; collagen I, 1:300; E‐cadherin, 1:500; Smad2/3, 1:300; Cleaved caspase‐1, 1:250; IL‐1β, 1:150), after that the corresponding fluorescent secondary antibodies, FITC and DAPI (Servicebio, Wuhan, China) were incubated with the samples for 1 h, 30 min, and 10 min, respectively. Similarly, the fluorescence signals were captured using a confocal laser microscope, whereas the fluorescence intensity determination of one protein selected an identical parameter.