Alexa fluor 488 labeled goat anti rabbit igg

All FFPE human skin biopsy samples and murine ear skin samples were sectioned into 2 and 4-μm-thick slides, respectively, and subsequently undergone hematoxylin-eosin (HE) staining. Human FFPE skin samples were stained immunohistologically with anti-human CD8 and anti-human CD4 monoclonal antibodies (clone C8/144B and 4B12, respectively, Nichirei Biosciences) using an automatic slide stainer according to the manufacturer’s instructions. The numbers of epidermal-infiltrating cells per sample (magnification, ×400) were counted. Murine ear FFPE samples were stained immunohistochemically with primary anti-CD3 (clone SP7, diluted 1:100, Abcam) and anti-CD8a (clone 4SM15, diluted 1:400, eBioscience) monoclonal antibodies, fluorescent-labeled secondary antibodies (Alexa Fluor^® 488-labeled goat anti-rabbit IgG, and Alexa Fluor^® 555-labeled goat anti-rat IgG, Abcam), and 4′,6-diamidino-2-phenylindole (DAPI) to detect the nucleus, by standard immunohistochemical staining techniques. A fluorescence microscope (BZ-X700, Keyence) was used for observation and to count the number of infiltrating cells per sample (magnification, ×400).

For cell immunofluorescent staining, the cells were seeded in a 12-well plate. After 24 h, the cells were fixed with 4% paraformaldehyde followed by a permeabilization procedure in 0.5% TritonX-100 (Sorlabio, Beijing, China) for 10 min. The cells were blocked with 10% horse serum (Solarbio, Beijing, Chain) for 1 h at room temperature. Primary antibodies against goal proteins were then incubated at 4 °C overnight. Subsequently, Alexa Fluor^® 488 labeled Goat Anti-Rabbit IgG (Abcam, Cambridge, UK) was used to specifically bind to the primary antibody, DAPI (Sorlabio, Beijing, China) was used for nuclei staining and the pictures were taken using a confocal microscope (ZEISS, Jena, German). For histoimmunofluoresence, the skin samples in anagen were cut into 7 µm sections with a freezing microtome (Leica, Nussloch, Germany). The slides were fixed with 4% paraformaldehyde; the subsequent process was as described in the cell immunofluorescence section. The following information of primary antibodies was used: α-SMA (Abcam, Cambridge, UK, VIM (Bioss, Beijing, China), and ITGA6 (Proteintech, Wuhan, China).

SCC cells grown on a glass-bottom culture dish (Life Technologies) were fixed in 4% paraformaldehyde solution. Primary antibody was rabbit anti-cIAP1 IgG (2 μg/ml; Abcam). Alexa Fluor 488-labeled goat anti-rabbit IgG was the secondary antibody (2 μg/ml; Abcam). After counterstain with 4',6-diamidino-2-phenylindole (DAPI), cells were observed under a digital confocal microscopy (Leica Company, Wetzlar, Germany).