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Multiplex staining by sequential immunostaining and antibody removal on routine tissue sections

By Maddalena Maria Bolognesi, Marco Manzoni, Carla Rossana Scalia, Stefano Zannella, Francesca Maria Bosisio, Mario Faretta, Giorgio Cattoretti

Posted 11 Jun 2017
bioRxiv DOI: 10.1101/148742 (published DOI: 10.1369/0022155417719419)

Multiplexing (mplx), labeling for multiple immunostains the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labelled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulphide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than thirty different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Mplx on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory and normal cells.

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