Rnascope antigen retrieval reagent

Formalin fixed, paraffin embedded (FFPE) tissue sections (5 µm thickness) from heart and skeletal muscle tissues selected from fish with HSMI, were mounted using Superfrost plus (Thermo Fisher Scientific) slides. Sections were baked at 60°C for 2 hrs in HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) prior to deparaffinization with absolute ethanol (100%) and fresh xylene. Initial blocking was done with hydrogen peroxide (Advanced Cell Diagnostics) for 10 min at room temperature (RT). RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) was used for 15 min at 99°C and slides were further incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven following manufacturer guidelines. Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA) was used to make hydrophobic barrier around tissue areas over slides for further probe hybridization procedures.

FFPE sections were sliced with 5 µm thickness from tissue samples and mounted on Superfrost plus (Thermo Fisher Scientific) slides. Slides were baked at 60°C for 2 h in a HybEZ™ II oven (Advanced Cell Diagnostics, catalog #321720) followed by deparaffinization with 100% ethanol and fresh xylene baths. Samples were pretreated with hydrogen peroxide for 10 min at RT, boiled with RNAscope antigen retrieval reagent (Advanced Cell Diagnostics, catalog #322000) for 15 min at 99°C, and then incubated with RNAscope protease plus reagent for 15 min at 40°C in the HybEZ™ II oven. Hydrophobic barrier was made around the tissue section using Immedge hydrophobic barrier pen (Vector Laboratories, Burlingame, CA).