fISH of murine EPOR (RNAscope Probe-Mm-Epor) was performed, using the RNAscope Multiplex Fluorescent reagent kit v2 from Advanced Cell Diagnostics and the fluorophore Opal 520 from PerkinElmer. Positive and negative control probes (RNAscope 3-plex positive and negative control probes) were always run in parallel.

Fresh frozen tissue was postfixed for 30 min at 4°C in 4% paraformaldehyde prepared in 0.15 m Na-phosphate buffer (pH 7.4), treated 10 min with hydrogen peroxide at RT, target retrieval for 10 min at 85°C (RNAscope Target Retrieval reagent) and protease treatment with Protease Plus for 30 min at 40°C. Probes were hybridized for 2 h at 40°C. Slides were washed in Tris-Triton buffer, pH 7.4 followed by amplification steps (RNAscope Amp1) and signal development (HRP-C1 + fluorophore 1). Finally, slides were incubated for 3 min in DAPI and coverslipped.

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