Rnascope 2.5 hd duplex assay kits

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.

RNA was visualized using RNAScope Multiplex Fluorescent Reagent kit v2 Assay, RNAScope 2.5 HD Detection Reagent – RED and RNAScope 2.5 HD Duplex Assay kits (Advanced Cell Diagnostics, ACDBio) using probes designed by Advanced Cell Diagnostics for ANKRD1, MYH6, NPPA, NPPB, CD163, DCN, POSTN, PLA2G2A, CCL2, PCOLCE2, ELN and RGS5 (ref. ^{77 (link)}). Samples were fixed for 24 h at 4 °C in 10% neutral buffered formalin. Samples were washed in 1× PBS, equilibrated in 30% sucrose, embedded in OCT medium (Sakura Finetek) and stored at −80 °C (fluorescence) or washed in 1× PBS, dehydrated in ethanol and embedded in paraffin (red and duplex). OCT-embedded sections were cut at 12 μm and paraffin-embedded sections were cut at 8 μm. Fluorescent images were collected using a Zeiss LSM 700 laser scanning confocal microscope. Chromogenic/brightfield images were acquired using a Zeiss Axioscan Z1 automated slide scanner. Image processing was performed using Zen Blue and Zen Black (Zeiss), FIJI/ImageJ^{78 (link),79 (link)} and Photoshop (Adobe). The following RNAScope probes produced by ACDBio were utilized: ANKRD1 (524241), MYH6 (555381), NPPA (531281), NPPB (448511), CD163 (417061), DCN (589521), POSTN (409181), PLA2G2A (581101), CCL2 (423811), PCOLCE2 (566861), RGS5 (533421), ELN (408261), ACKR1 (525131), BTNL9 (430351) and CCL21 (474371).