3 3 diaminobenzidine (dab)

Slices of tissues were subjected to antigen retrieval by heating in a microwave oven in 10 mmol/L citrate buffer for 3 min. Then, sections were incubated with primary antibodies against Ki-67 (ab16667, Abcam), FOXQ1 (MA5-17078, ThermoFisher) and CD133 (ab284389, Abcam) at 4°C overnight. Afterward, slices were incubated with poly-peroxidase-anti-mouse/rabbit IgG and detected using DAB (Bioss, Beijing, China). Hematoxylin-based reagents were used to create the immunohistochemistry response, which was subsequently seen under a microscope.

The biopsy specimens obtained from the UV-exposed back skin were dissected immediately following the measurement of footpad thicknesses. The tissues were fixed in 4% formaldehyde (Guangzhou Chemical Reagent Factory, Guangzhou, China) solution in phosphate-buffered saline (PBS) and cut using a Leica RM2235 microtome (Leica Microsystems GmbH- Wetzlar, Germany) into sections of 4 µm. The sections were then deparaffinized in xylene (Guangzhou Chemical Reagent Factory), and hydrated in a series of ethanol and distilled water. Endogenous peroxidase was eliminated by incubating the sectioned tissues in 5% H₂O₂ in methanol for 30 min. Non-specific staining was blocked by incubation in 5% normal mouse serum in PBS for 30 min at 37°C. The sections were then incubated overnight at 4°C with primary antibodies, including mouse anti-CD207/Langerin, anti-CD80 and anti-CD86 antibodies (1:200). Following rinsing three times for 5 min with PBS, the sections were incubated with HRP-conjugated anti-mouse IgG (1:2,000) for 1 h. The sections were rinsed with PBS twice for 10 min, following which the slides were developed using 3,3′-diaminobenzidine (Beijing Biosynthesis Biotechnology). The sections were examined using a Nikon Eclipse TE2000-U light microscope (Nikon Corporation, Tokyo, Japan), and images captured with a digital camera.