Multiple biotin-avidin amplification for multiple immunostaining

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