Streptavidin hrp antibody

Proteome profiler-mouse cytokine array panel A (R&D Systems) was subjected to analysis. All protocols were performed according to the manufacturer's instructions. Briefly, 1 ml of freshly collected T cell conditional medium was applied to pre-blocked cytokine array membrane. After 1 h incubation, detection antibody cocktail (R&D systems) was added and incubated with the membrane overnight at 4 °C. Streptavidin-HRP antibody (R&D systems) was used as the secondary antibody. The signals were detected by Las4000 X-ray device (GE Healthcare) and the pixel density was analyzed by Las4000 software (GE Healthcare).

96-well microplates were coated by 0.2 μg antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. Total proteins extracted from TA muscles were applied to the microplates. Biotinylated goat antibodies against IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience) were applied as detection antibodies, respectively. Streptavidin-HRP antibody (R&D systems) was used as the secondary antibody. The signals were collected by microplate reader (Thermo MK3, Thermo Fisher) and quantified by comparison with standard solution containing recombinant mouse IL-1α (R&D systems), IL-13 (R&D systems), IFN-γ (eBioscience) and TNF-α (eBioscience), respectively. The standard curves were made according to the manufacturer's instructions.