Multiple Iteractive Labeling by Antibody Neodeposition (MILAN)

What's new in protocol Version 5: an expanded troubleshooting section, more validated antibodies) Multiplexing, labeling for multiple immunostains the very same cell or tissue section in situ, is of considerable interest.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 and detail here a method based on common primary and secondary antibodies, diffusely available uorescent image scanners and routinely processed tissue sections \(FFPE).It entails rounds of fourcolor indirect immuno uorescence, image acquisition and removal \(stripping) of the antibodies, before another stain is applied.The images are digitally registered and the auto uorescence is subtracted.Removal of antibodies is accomplished by disulphide cleavage.In excess of 50 different antibody stains can be applied to one single section from routinely xed and embedded tissue.This method requires a modest investment in hardware and materials and uses freeware image analysis software.