Multiplexed Imaging of Tissue Sections

For IMC histology, tissue sections were de-paraffinized and rehydrated, and antigen retrieval was performed in pH 8, 1 mM EDTA buffer at 96 °C for 20 min. Sections were cooled at room temperature and rinsed in tap water and TBS (20 mM Tris with 500 mM NaCl, pH 7.5). Tissue was blocked for 3 h at room temperature with 0.3% BSA, 25% FBS and 0.1 mg/mL FC receptor binding inhibitor in TBS-T (TBS + 0.05% Tween-20). All antibodies (Additional file 1: Table S2) were diluted in 0.3% BSA in TBS-T and applied to the tissue for overnight incubation at 4 °C. Sections were then rinsed in TBS-T and TBS, and counterstained with 125 nM Maxpar® Intercalator-Ir (Fluidigm) in PBS for 1 h at room temperature. Sections were rinsed in TBS-T, TBS, and two washes of distilled water before air-drying at room temperature. Antibody-labeled tissue areas (1000 × 1000 μm) were raster-ablated using a Hyperion™ Laser Scanning Module (Fluidigm) with a 1 μm diameter spot size at 200 Hz. This process was coupled to a Helios™ Mass Cytometer (Fluidigm) for lanthanide metal detection [43 (link)]. Images for each antibody channel were acquired on CyTOF Software (Fluidigm, version 6.7). MCD Viewer (Fluidigm, version 1.0) was used to export raw 16-bit tiff images for computational analyses on histoCAT (version 1.75) [36 (link)]. For visualization purposes, images were processed in MCD Viewer and ImageJ [38 (link)].