Four µm-thick sections were cut from each block of samples CLN1-CLN6 with a microtome and used for staining with a panel of 26 antibodies targeting the main immune, stromal and epithelial cell populations of the gastrointestinal tract (Supplementary Table 2). The optimal dilution of each antibody in the panel was identified by staining and ablating FFPE appendix sections. The resulting images were reviewed by a mucosal immunologist (J.S.) and the dilution giving the best signal to background ratio was selected for each antibody (Supplementary Table 2). To perform the staining for IMC, slides were dewaxed after a one-hour incubation at 60°C, rehydrated and heat-induced antigen retrieval was performed with a pressure cooker in Antigen Retrieval Reagent-Basic (R&D Systems). Slides were incubated in a 10% BSA (Sigma), 0.1% Tween (Sigma), and 2% Kiovig (Shire Pharmaceuticals) Superblock Blocking Buffer (Thermo Fisher) blocking solution at room temperature for two hours. Each antibody was added to a primary antibody mix at the selected concentration in blocking solution and incubated overnight at 4°C. After two washes in PBS and PBS-0.1% Tween, the slides were treated with the DNA intercalator Cell-ID™ Intercalator-Ir (Fluidigm) (containing the two iridium isotopes 191Ir and 193Ir) 1.25 mM in a PBS solution. After a 30-minute incubation, the slides were washed once in PBS and once in MilliQ water and air-dried. The stained slides were then loaded in the Hyperion Imaging System (Fluidigm) imaging module to obtain light-contrast high resolution images of approximately four mm2. These images were used to select the ROI in each slide. For CLN1-CLN6, 1 mm2 ROIs were selected to contain the full thickness of the colon mucosa, with epithelial crypts in longitudinal orientation. ROIs were ablated at a o µm/pixel resolution and 200 Hz frequency. Twenty-eight images from 26 antibodies (Supplementary Table 2) and two DNA intercalators were obtained from the raw .txt files of the ablated regions in CLN1-CLN6 using the data extraction process.

.mcd files with one or more ablated ROIs channel_metadata.csv = Assigns each metal/channel to the corresponding marker raw_file_metadata.csv = Assigns each sample to an ROI and a .mcd file