Simple generation of cleavable labels for multiplexed imaging†

Abstract

The most common methods for multiplexed immunohistochemistry rely on cyclic procedures, whereby cells or tissues are repeatedly stained, imaged, and regenerated. Here, we present a simple and inexpensive approach for amine-targeted labeling of antibodies using a linker that can be easily cleaved by a mild reducing agent. This method requires only inexpensive and readily-available reagents, and can be carried out without synthetic experience in a simple one-pot reaction. We demonstrate the applicability of this approach by performing repeated staining-imaging-removal cycles on isolated cells and tissue sections, finding that over 94% of the labels can be removed within 30 minutes using only the gentle application of reducing agent, increasing up to 99% by extending the incubation duration to 1 hour. By providing a convenient way to introduce cleavable linkers, our method simplifies methodologies such as high-content imaging or multiplexed immunohistochemistry.