The AEC coloring system consists of two items: a concentrated AEC stock solution and a concentrated Substrate buffer. Mix 1 ml Substrate buffer (10x) thoroughly with 4.2 ml 70% ethanol and 4.8 ml demineralized water to reach a final concentration of substrate buffer (1x) in 30% ethanol. Add 660 μl AEC stock solution* (toxic: use a fume hood) and mix thoroughly. After mixing, the solution should be clear. This amount is sufficient for 1 ELISPOT plate (96 determinations). The system provides an effective substrate for the streptavidin-HRP conjugate (CT353) generating red-colored spots on PVDF membranes. Upon ordering, you will receive one vial AEC stock solution (7 ml, to be stored at -20ºC upon arrival) and 10 ml Substrate buffer for 960 determinations.