Multiple biotin-avidin amplification for multiple immunostaining

A multiple fluorescence immunostaining method is described, based on the us e of multiple fluorochrome-avidin/streptavidin conjugates. Key factors are (a) stabilization of each (preceding) biotin-avidin chain and associated av idin blocking step with formaldehyde and (b) application of two or three (p ossibly more) fluorochrome-labeled avidins/streptavidins in decreasing orde r of visual sensitivity. Under the conditions described, no cross-talk was detected, and both signal and background levels were comparable to correspo nding single immunostaining. Enzyme-linked immunosorbent assay experiments confirmed effective inhibition of competition-displacement of previously bo und avidin after the blocking-stabilization procedure used.