Figure 1.

Schematic overview of the currently available methods for multiplexed immunohistochemistry (IHC). (A) Currently, the most common approach for multiplexed IHC makes use of fluorescently labelled probes, which are either directly coupled to the primary antibody or indirectly provided by a secondary antibody, that are detected in a cyclic fashion consisting of a staining protocol, followed by tissue imaging and signal removal. (B) In contrast to cyclic methods, single-step spectral methods detect all dyes in the tissue simultaneously: these can either be provided by directly labelled antibodies that are all simultaneously present in the tissue section or by the cyclic generation of TSA precipitates which are subsequently spectrally unmixed in a single imaging step. (C) Antibodies can also be detected by covalently linked nucleotide labels to which fluorescently labelled probes are hybridized in a cyclic fashion for which each cycle gets imaged. (D) Non-fluorescent mIHC methods involve the cyclic generation of chromogenic substrates that are washed away following an imaging step in between each cycle. (E) For imaging mass cytometry (IMC), antibodies are labelled with metal isotopes which are detected by the local vaporization of the metal ions by a UV laser, following which the present isotopes are resolved using atomic spectrometry. (F) Finally, nucleotide labelled antibodies can be detected by removing the nucleotide labels from the antibodies using a laser beam, following which the nucleotides that were collected from a precise region of interest are sequenced to quantify the amount of available proteins in that region.