Horseradish peroxidase conjugated streptavidin

Supernatants, obtained from cultured human dermal TCs and Fbs, were analysed with cytokine antibody array (Human Cytokine Antibody Array V, RayBiotech Inc., Shanghai, China). This study was performed by Kangchen Bio-tech Company (Shanghai, China). Briefly, cytokine array membranes were blocked in blocking buffer for 30 min. and then incubated with samples at room temperature for 1–2 hrs. Samples were then decanted, and membranes were washed. The membranes were incubated with diluted biotin-conjugated antibodies (Cusabio, DE, USA) at room temperature for 1–2 hrs. After the membranes were washed again, 1000-folds diluted horseradish peroxidase-conjugated streptavidin (Proteintech, Chicago, IL, USA) was added and incubation was continued for 2 hrs at room temperature. The membranes were then washed thoroughly and exposed to detection buffer (Raybiotech, Atlanta, GA, USA) in the dark, before being exposed to X-ray film. By comparing the signal intensities, relative expression levels of cytokines could be obtained. The intensities of signals were quantified by densitometry. Biotin-conjugated immunoglobulin G (Cusabio) served as a positive control at six spots to normalize the results from different membranes being compared.