Calcein pi staining kit

Skin lesions were fixed in paraformaldehyde for at least 48 h and embedded in paraffin. Sections (5 µm) were dewaxed using graded xylene and ethanol. Hematoxylin and eosin (H&E) as well as Masson’s trichrome (MT) staining was conducted according to the manufacturer’s instructions. CD31 (1:500; Abcam, Cambridge, UK), alpha smooth muscle actin (α-SMA, 1:200; Abcam) and perilipin A (1:800; Abcam) were used for immunohistochemical assays. For immunofluorescence assay, sections were incubated with following antibodies: F4/80 (1:100; Abcam), GFP (1:250; Abcam), Tdtomato (1:250; Abcam), Sox9 (1:200; Abcam) and then goat anti-chicken secondary antibody, Alexa Fluor 488 (1:500; Thermo Fisher Scientific, MA, USA) and goat anti-rabbit secondary antibody, Alexa Fluor 555 (1:500; Thermo Fisher Scientific). The cell viability was tested according to Calcein/PI staining kit (Beyotime Biotechnology, Shanghai, China). Images were obtained using an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) or fluorescence microscope (Imager Z2, Carl Zeiss) and analyzed using the ImageJ software. Number of hair follicles and sebaceous glands was count manually on the samples with H&E staining.

The Calcein/PI staining kit (Beyotime, Shanghai, China) was used to assess the pyroptotic cell death of cells. After coincubation with Calcein/PI staining kit (1 μl/ml) for 30 min at 37 °C, followed by resuspension in PBS. After that, the cells visualized using fluorescence microscopy (Leica, Bensheim, Germany).