Table 1.

Comparison of method parameters for each workflow. Workflows were done in sequence using manufacturer’s protocol method parameters. The dewaxing procedures were the same and are described in the “Materials and methods” section. Separate antigen retrieval was performed for MALDI-MSI of N-glycans and ECM peptides to expose the correct sites for protease access

	Initial heating time (h)	Initial heating temperature	Antigen buffer (pH)	Antigen retrieval device	Antigen retrieval temperature	Antigen retrieval time (min)	Type of antibody
MALDI-MSI: N-Glycans	1	60 °C	Citraconic, 10 mM pH 3.0 prepared in house	Vegetable steamer	95 °C	25**	NA
MALDI-MSI: ECM peptides	1	60 °C	Tris, 10 mM pH 9.0 prepared in house	Vegetable steamer	95 °C	25**	NA
AmberGen MALDI HiPLEX-IHC	2	60 °C	Tris-EDTA, pH 9.0 commercial preparation	Water bath	95 °C	30	Photocleavable mass-tags
GeoMx Digital Spatial Profiler	4	70 °C	Tris-EDTA, pH 9.0 commercial preparation	Rice cooker	95 °C	15	Fluorescent and oligo-conjugated
Imaging Mass Cytometry (IMC)	1	60 °C	Tris-EDTA, pH 9.0 commercial preparation	Decloaker	95 °C*	20	Metal-conjugated

^**Time was reduced to 15 min for the breast TMA to avoid tissue loss