Table 1.

IHC and IF multiplexing techniques.

Method	Process	Advantages	Disadvantages	References
MICSSS (IHC)	Multiple staining rounds; AEC removal AEC with organic solvent-based destaining buffer; imaging	No limitation by the number of different antibody species No company-specific reagents/devices are needed	Time intensive Limited to 10 staining rounds	[29]
SIMPLE (IHC)	Multiple staining rounds; AEC removal with organic solvent-based destaining buffer; imaging	No limitation by the number of different antibody species No company-specific reagents/devices are needed	Time intensive Limited to 5 rounds of staining without loss of tissue antigenicity	[28]
Opal mIHC (IF)	sequential staining with AB tagged with TSA conjungated fluorescence molecules, AB removal by heat-treated antibody stripping; imaging	No limitation by the number of different antibody species up to 7 markers	Time intensive limited by the number of fluorochromes	[30]
In silico multiplexing workflow (IF)	Multiple staining rounds; Dye inactivation by bleaching with alkaline solution + H2O2; imaging	No limitation by the number of different antibody species No company-specific reagents/devices are needed	Each round of staining may take at least 24 h depending on the antibodies and the tissue	[31]
t-Cycif (IF)	Multiple staining rounds (like MxIF); bleaching by hydrogen peroxide, intense light and high pH; imaging	Background noise decreases with cycle number due to multiple rounds of fluorophore bleaching No limitation by the number of different antibody species No company-specific reagents/devices are needed	Relatively slow (each cycle 6–8 h, most time consuming is the imaging) after 10 cycles, 2–45% loss of cells	[32]
MxIF (IF)	Multiple staining rounds; Alkaline oxidation chemistry was developed that eliminates cyanine-based dye fluorescence within 15 min; imaging	No limitation by the number of different antibody species No company-specific reagents/devices are needed Removal of fluorescence dye within 15 min Up to 60 biomarkers	relatively slow due to scanning times	[33]
MELC (IF)	Multiple automatic staining rounds; during each cycle the sample is incubated with one or more tags and imaged before bleaching by soft multi-wavelength excitation	Automated cycles of fluorescent staining, imaging and photobleaching No limitation by the number of different antibody species	Bleaching/acquisition can be applied only to one field of view Special devices are needed	[35]
CODEX (IF)	Antibodies conjugated to a CODEX barcode; visualized by the binding of highly specific corresponding dye-labeled CODEX reporter	No limitation by the number of different antibody species -> no secondary antibodies Fast, each round of extension and bleaching (10 min) Up to 35 rounds with 3 markers	Special devices and reagents are needed	[34]
NanoString (IF)	Antibodies conjugated to a barcode; visualized by the binding of highly specific corresponding dye-labeled reporter	Up to 40 markers No autofluorescence and spectral overlap	Limited number of regions of interest Special devices and reagents are needed	[26]

AB, antibody; AEC, 3-amino-9-ethylcarbazole; CODEX, co detection by indexing; IF, immunofluorescence; IHC, immunohistochemistry; MELC, multi-epitope-ligand cartography; MICSSS, multiplexed immunohistochemical consecutive staining on a single slide; SIMPLE, sequential immunoperoxidase labelling and erasing method; TSA, tyramide signal amplification system; t-Cycif, tissue-based cyclic immunofluorescence.