F4 80 rabbit pab

Healthy and naturally infected New Zealand rabbit toe skins were fixed overnight with a 4% paraformaldehyde fixative (Biosharp, Guangzhou, China). The paraffin-embedded skin samples were sectioned to 5 μm, dried in an oven at 37°C for 24 h, deparaffinized, immersed in 0.1 M sodium citrate for 15 min for antigen retrieval, and washed three times with phosphate-buffered saline (PBS). The 5% goat serum was used to block at room temperature for 2 h, and then the primary antibody, F4/80 Rabbit pAb (1:200) (ABclonal, Wuhan, China), was incubated overnight at 4°C and washed three times with PBS, followed by the secondary antibody, FITC Goat Anti-Rabbit IgG (H+L) (1:400) (ABclonal, Wuhan, China), incubated for 45 min at room temperature. After washing four times, DAPI (Solarbio, Beijing, China) was used to incubate for 5 min at room temperature. Following mounting, tissue sections were examined and captured on a camera using a digital slice microscope [OLYMPUS, VS120-S6-W (BX61VS), Japan].

Paraffin sections of mouse AD model skin were prepared as previously described. Stained with hematoxylin and eosin (H&E) to determine the thickening and cellular infiltration of the skin. Furthermore, the immunofluorescence procedure was performed in the same way as shown in macrophage localization. F4/80 Rabbit pAb (1:200) (ABclonal, Wuhan, China) was employed as the primary antibody and FITC Goat Anti-Rabbit IgG (H+L) (1:400) (ABclonal, Wuhan, China) as the secondary antibody to observe macrophage infiltration in experimental mice. A digital section microscope was used to monitor and photograph both H&E and F4/80 practical sections.