Hematoxylin

A group of mice was sacrificed on Day 6 and their skin samples were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and stored at RT until used. Paraffin sections (8 μm) were cut using a paraffin slicer (Leica, Solmas, Germany) and stained with H&E (Beyotimes, Shanghai, China). Images of H&E staining were acquired using an eclipse upright optical microscope digital camera (Nikon, Tokyo, Japan). Two random positions from each mouse were selected to measure the epidermal thickness by Image J software (version 1.8.0, Maryland). Baker’s scores (29 (link), 29 (link)) were used to analyze the pathological severity of the skin. CD11c positive cells were stained with biotin-rat anti-mouse CD11c antibody (1:100, Biolegend, California) for 1 h, followed by streptavidin-HRP antibody (1:800, Yeasen, Shanghai, China) incubation for 40 min. Sections were developed with DAB (Vector Laboratories, California) and counter-stained with hematoxylin (Phygene, Shanghai, China) before visualization under the microscope (Nikon). CD11c positive cells (30 (link)) were counted in 5 microscopic fields under 200X magnification and expressed as cells/field, and mean ± SEM.

Cell type and distribution were determined by H&E staining and immunohistochemical staining. The specific experimental steps refer to the previous literature of our team (28 (link)). Primary and secondary antibodies are listed in Table S2. For antigen retrieval, the paraffin tissue sections were immersed in citrate buffer (pH 6.0), and placed in a pressure cooker until steam was generated for 3 min. To block non-specific peroxidase, a 3% hydrogen peroxide and methanol solution was applied to tissue sections for 10 min, following incubation in goat serum (Solarbio Life Science, Beijing, China, S9070) for 30 min. Tissue sections were then incubated with diluted primary antibody (Table S2) at 4°C overnight and washed twice with PBS. Fluorescent dye-labeled or HRP-labeled secondary antibodies were applied to the tissue sections for 30 min, followed by three PBS washes. The nuclei were counterstained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Solarbio Life Science, Beijing, China, C0065) or DAB (Dako, Glostrup, Denmark, 20052898) and hematoxylin (Scientific Phygene, Fuzhou, China, PH1464). Images of all sections were captured using a Leica DM2500 microscope.

Frozen slices were prepared for immunohistochemistry by dehydrating in 30% sucrose-PBS solution for 48 h at 4°C. Skin samples were embedded in optimal cutting temperature compound (OCT, Sakura Finetek, California, USA) and frozen quickly overnight at −80°C. Eight μm sections were cut by using a microtome (Leica) and stored at −20°C until used. Before staining with antibodies, 5% goat serum was used for blocking, and then tissues were dehydrated with 75–95% ethanol. Innate immune cells were stained with biotin-rat anti-mouse Gr-1 (1:200, BioLegend, California, USA), CD11c (1:100, BioLegend), or F4/80 (1:100, BioLegend) antibodies for 1 h, followed by an incubation with streptavidin-HRP (1:800, Yeasen, Shanghai, China) for 40 min. DAB solution (Vector Laboratories, California, USA) was used for color development and hematoxylin (Phygene, Shanghai, China) was used for counterstaining before visualization under the microscope (Leica). F4/80 and Ly6G positive cells in each mm² were calculated under an optical microscope.