Rnascope probe

Flash-frozen LV samples were fixed for 24 hours at 4 °C in 10% neutral buffered formalin, washed in 1× PBS and embedded in paraffin. Paraffin-embedded sections were cut at an 8-μm thickness using a microtome. RNA in situ hybridization was performed using the RNAScope^{69 (link)} Multiplex Fluorescent Reagent Kit version 2 assay and RNAScope 2.5 HD Detection Reagent as per the protocol—RED and RNAScope 2.5 HD Duplex Assay Kits (Advanced Cell Diagnostics) using probes designed by Advanced Cell Diagnostics. Fluorescent images were collected using a Zeiss LSM 700 laser scanning confocal microscope. The following RNAScope probes from Advanced Cell Diagnostics were used: MYH6 (555381), NPPA (531281), CD163 (417061), POSTN (409181), PLAUR and RUNX1. Chromogenic/bright-field/fluorescent images were acquired using a Zeiss Axioscan Z1 automated slide scanner. Image processing was performed using Zen Blue and Zen Black (Zeiss). Positive cells were counted using either of two approaches: (1) for fluorescent images, the number of positive cells counted per ×10 field; or (2) for chromogenic images, the number of positive interstitial cells / total number of interstitial nuclei per ×10 field. For CD163 and POSTN, donor and DCM samples were used from our previously published study and re-quantified as per the above approach to ensure comparability.

All relevant data are available from the authors and/or are included in the manuscript or supplementary information. The RNAscope probes used in this study were designed by Advanced Cell Diagnostics, Newark, CA).

RNAscope probes were obtained from Advanced Cell Diagnostics. In situ hybridization was performed following RNAscope Multiplex Fluorescent Reagent Kit v2 user manual (document #323100-USM). Briefly, perfusion with PBS, kidneys were harvested and fixed at 4°C in 4% PFA in PBS for 1 hour, followed by overnight at 4°C in 30% sucrose. Kidneys then were embedded in OCT in molds floated on a dry ice/ethanol bath. Cryosections (10 µm) were fixed in 4% PFA at 4°C overnight before the standard pretreatment steps for fixed frozen tissue sample in the protocol. Signal amplification after the RNAscope Multiplex fluorescent v2 assay was performed with TSA plus fluorophores as recommended and Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific). Complete details of RNAscope probes used here are provided in Key Resource Table.