The in situ RNA amplification and labeling process were performed at ACD (Newark, CA) by the Pharma Assay Services group using the RNAscope LS Multiplex Reagent Kit along with the 4-Plex Ancillary Kit according to the manufacturer’s instructions on a Leica Bond Rx autostainer. Probes were assigned to the following channel and fluorophore configuration: Mm-Ins2-C1 (Opal 520)/Mm-Gcg-C2 (Opal 570)/Mm-Sst-C3 (Opal 620)/Mm-Malat1-C4 (pooled probe, Opal 690). Using the positive and negative control probes, optimization was performed to establish the following pretreatment conditions: 15 min ER2 at 88°C and 15 min protease III at 40°C.

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