GSM8381820: PR79_1_WTA | 054 | Trp; Homo sapiens; OTHER
Adult human heart samples were harvested between January 2018 and April 2021 and kept in paraffin blocks after formalin fixation. Control samples were obtained from autopsy cases with no history of HF or extracted from the left ventricle (LV) of the patients with normal LV function while receiving a heart transplant or a heart-lung transplan for isolated right ventricular failure or other reasons. Failing hearts were obtained from explanted hearts of recipients receiving a heart transplant, from excisional biopsy of patients receiving a LV assisant device, from myomectomy for obstructive HCMP, and from autopsy. Formalin-fixed, paraffin-embedded sections were deparaffinized, went through antigen retrieval procedures, and then incubated with human WTA UV-cleavable RNA probes. They were co-stained with fluorescently conjugated antibody antibody morphologic markers against Troponin I, Vimentin, and CD31. After preparing tissue, bake slides with mounted sections in 60C drying oven for 30min to 3 hours. Deparaffinize and rehydrate FFPE tissue sections for 35 minutes. Target retriveal were performed for 25 minutes After exposing RNA targets, add 200 uL of hybridization solution made by manufacturer's buffer(Buffer R, Atlas Probe Mix, Custom assay1, Custom assay2, DEPC-treated H2O) in the hybridization chamber. Gently apply a Grace Bio-Labs HybriSlip and incubate at 37C for obernight at hybridization oven. Stringent wash was performed using 4X SSC with formamide and 2x SSC to remove off-target probes. Load prepared slides in to the GeoMX DSP instrument. Slides are scanned to capture fluorescent images used to select regions of interest(ROIs). The instrument collects UV-cleaved oligos from the ROIs into the wells of a collection plate.