Fluorophore conjugated streptavidin

This assay was accomplished with Proteome Profiler Mouse Cytokine Array Panel A kit (Cat#ARY006, R&D system)^{35 (link)}. For each membrane, 200 µl of protein solution was mixed with 100 µl of sample buffer (array buffer 4) and 1.2 ml of block buffer (array buffer 6), then added with 15 µl of reconstituted Mouse Cytokine Array Panel A Detection Antibody Cocktail and incubated at room temperature for 1 h. The array membrane was incubated with block buffer (array buffer 6) for 2 h on a rocking platform shaker in the meantime, and then the block buffer was aspirated, the prepared sample/antibody mixture was added onto the membrane and incubated overnight at 4 °C on a rocking platform shaker. The membrane was washed with 20 ml of 1× wash buffer for 10 min on a rocking platform shaker for three time and rinsed with deionized water once, then was probed with Fluorophore-conjugated streptavidin (1:5,000 dilution, Cat#926-32230, Li-Cor) at room temperature for 30 min on a rocking platform shaker, washed with wash buffer for three times and rinsed with deionized water once again as in above steps. Antibody-antigen complexes were visualized using Odyssey Detection (Li-Cor, Serial No. ODY-2329) at 800 nm wavelengths. The densities of the spots were analyzed by Image J software.