Fitc tyramide

The skin lesion tissues were fixed, paraffin-embedded, sliced sections (thickness 3 μm), and dewaxed. Then, microwave oven antigen repair in citrate buffer, endogenous peroxidase was blocked by 3% hydrogen peroxide, serum was blocked. Next, add F4/80 antibody (1:100, cat no: 28463-1-AP, Proteintech, Wuhan) at 4℃ overnight, dropwise add HRP-labeled secondary antibody (1:100, cat no: GB23303, Servicebio, Wuhan) and incubate at room temperature for 30 min, add FITC-Tyramide (1:500, cat no: G1222, Servicebio, Wuhan) and incubate at room temperature for 10 min. Sections were submerged into citrate buffer microwaved for 10 min, followed by the addition of antibodies against iNOS (1:100, cat no: GB11119, Servicebio, Wuhan), CD206 (1:100, cat no: GB113497, Servicebio, Wuhan), and Notch1 (1:100, cat no: 11976R, Bioss, Beijing) incubated overnight at 4℃, and dropwise addition of fluorescent secondary antibody (CY3 labeled goat anti-rabbit, cat no: GB21303, Servicebio, Wuhan) was incubated at 37℃ for 30 min. Images were captured using digital scanning and browsing software (OlyVIA, OLYMPUS, Japan) and the fluorescence intensity of all acquired images was measured using the Image-J analysis system (National Institutes of Health, USA).