Hyperion (Imaging mass cytometry) Source Data

Primary antibodies were prepared carrier-free, except for YKL-40, which contained glycerol and was purified prior to metal conjugation using Amicon 10K Buffer Exchange Columns (EMD Millipore, UFC501096). Antibody concentrations were determined using a Nanodrop 2000c Spectrophotometer and adjusted to a final stock concentration of 0.5 mg/ml. All antibodies were conjugated following Standard BioTool's (formerly Fluidigm) Maxpar X8 protocol using Maxpar metal labeling kits (SBT, 201300).

Fixed, cryoprotected tissue was sectioned at 14μm thickness on a HM525NX cryostat (Fisher) at -15°C and mounted on Fisher Superfrost Plus slides, which were stored at -80°C until staining. We followed Standard BioTool's fresh frozen staining protocol, omitting the fixation step due to prior tissue fixation. Slides were incubated at 37°C for 5 minutes on a PCR machine, similar to the 10x Genomics Visium protocol, then washed in PBS three times for 5 minutes. After drawing and drying a hydrophobic barrier, sections were incubated with 3% BSA in PBS with 0.2% Triton X-100 for 45 minutes at room temperature, followed by overnight incubation at 4°C with the primary antibody cocktail diluted in 0.5% BSA/PBS with 0.2% Triton X-100 (details in Supplementary Table 29). The sections were washed in PBS with 0.2% Triton X-100 twice for 8 minutes, then incubated with iridium intercalator (1:100 in PBS, SBT, 201192A) for 30 minutes at room temperature.

Sections were washed twice in water for 5 minutes, air-dried, and then subjected to ablation. The Hyperion Imaging System (SBT) was tuned using the Hyperion Tuning Slide (SBT, 201088) prior to ablation, which was performed with an energy of four and a reference energy of zero in 1000x1000μm regions of interest, except for one acquisition at 1000x922 due to unexpected Argon gas depletion. Raw images are deposited along with metadata.