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文献和实验Dual- and Triple-Color Fluorospot
in several publications and reagents/kits for various cytokine combinations and species are commercially available. The most common protocol for fluorospot includes the use of biotinylated detection antibodies for one cytokine and an FITC-labeled detection antibody
, binds secreted analyte. After washing away any cells and unbound substances, a biotinylated polyclonal antibody specific for the chosen analyte is added to the wells. Following a wash to remove any unbound biotinylated antibody, alkaline-phosphatase
Immunofluorescent Staining of Mouse and Rat Leukocytes
cold wash buffer (PBS/0.1% NaN3/1.0% fetal bovine serum). Centrifuge at 350 x g for 5 min. Finally, resuspend cell pellet to a concentration of 2 x 107 cells/ml (i.e., 106 cells per 50 µl). Dilute primary mAbs (e.g., unconjugated, biotinylated
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