Simultaneous amplification of multiple immunofluorescence signals via cyclic staining of target molecules using mutually cross-adsorbed antibodies.

Amplification of immunofluorescence (IF) signals is becoming increasingly critical in cancer research and neuroscience. Recently, we put forward a new signal amplification technique, which we termed fluorescent signal amplification via cyclic staining of target molecules (FRACTAL). FRACTAL amplifies IF signals by repeatedly labeling target proteins with a pair of secondary antibodies that bind to each other. However, simultaneous amplification of multiple IF signals via FRACTAL has not yet been demonstrated because of cross-reactivity between the secondary antibodies. In this study, we show that mutual cross-adsorption between antibodies can eliminate all forms of cross-reactions between them, enabling simultaneous amplification of multiple IF signals. First, we show that a typical cross-adsorption process-in which an antibody binds to proteins with potential cross-reactivity with the antibody-cannot eliminate cross-reactions between antibodies in FRACTAL. Next, we show that all secondary antibodies used in FRACTAL need to be mutually cross-adsorbed to eliminate all forms of cross-reactivity, and then we demonstrate simultaneous amplification of multiple IF signals using these antibodies. Finally, we show that multiplexed FRACTAL can be applied to expansion microscopy to achieve higher fluorescence intensities after expansion. Multiplexed FRACTAL is a highly versatile tool for standard laboratories, as it amplifies multiple IF signals without the need for custom antibodies.

PICASSO allows ultra-multiplexed fluorescence imaging of spatially overlapping proteins without reference spectra measurements.

Ultra-multiplexed fluorescence imaging requires the use of spectrally overlapping fluorophores to label proteins and then to unmix the images of the fluorophores. However, doing this remains a challenge, especially in highly heterogeneous specimens, such as the brain, owing to the high degree of variation in the emission spectra of fluorophores in such specimens. Here, we propose PICASSO, which enables more than 15-color imaging of spatially overlapping proteins in a single imaging round without using any reference emission spectra. PICASSO requires an equal number of images and fluorophores, which enables such advanced multiplexed imaging, even with bandpass filter-based microscopy. We show that PICASSO can be used to achieve strong multiplexing capability in diverse applications. By combining PICASSO with cyclic immunofluorescence staining, we achieve 45-color imaging of the mouse brain in three cycles. PICASSO provides a tool for multiplexed imaging with high accessibility and accuracy for a broad range of researchers.

FRACTAL: Signal amplification of immunofluorescence via cyclic staining of target molecules.

In this article, we demonstrate fluorescent signal amplification via cyclic staining of target molecules (FRACTAL), a technique that can amplify the signal intensity of immunofluorescence staining more than nine-fold via simple cyclic staining of secondary antibodies. We also show that FRACTAL is compatible with four-color imaging and expansion microscopy imaging.