RT Journal Article SR Electronic T1 Multiplex staining by sequential immunostaining and antibody removal on routine tissue sections JF bioRxiv FD Cold Spring Harbor Laboratory SP 148742 DO 10.1101/148742 A1 Maddalena Maria Bolognesi A1 Marco Manzoni A1 Carla Rossana Scalia A1 Stefano Zannella A1 Francesca Maria Bosisio A1 Mario Faretta A1 Giorgio Cattoretti YR 2017 UL http://biorxiv.org/content/early/2017/06/11/148742.abstract AB 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.