Sample preparation is critical for successful staining of tissue with RNAscope® ISH methodology. RNAscope® manual assay can be used with FFPE (formalin-fixed, paraffin-embedded) tissue, cultured cells, fresh-frozen & or fixed-frozen tissues, or PBMC (Peripheral Blood Mononuclear cells). Please refer to the appropriate sample preparation guides as shown in the User Manual Selection Guide available at User manuals to learn about the sample preparation pretreatment methods for the detection assay and to ensure you have the RNAscope® Pretreatment reagents, before you start the assay.
FFPE Tissue specimens should be blocked into a thickness of 3 - 4 mm and fixed for 24 +/- 8 hours in 10% neutral-buffered formalin at room temperature. The fixed tissues should then be dehydrated in a graded series of ethanol and xylene, followed by infiltration by melted paraffin held at no more than 60°C. Specimens should be analyzed within 3 months of sectioning when stored at room in desiccant temperature (20-25 °C). Slides need to be air dried and baked at 60°C for 1-2 hours prior to the RNAscope® Assay. FFPE Tissue sections should be cut into sections of 5 +/-1μm. Use Fisher Scientific SuperFrost Plus Slides for all tissue types to avoid tissue loss. Tissue thickness for fixed frozen tissue should be between 7–15 µm and 10–20 µm for fresh frozen tissue.
We recommend optimizing conditions for the RNAscope Assay if tissue samples are not prepared according to the ACD recommended guideline of fixing samples in fresh 10% NBF for 16–32 hours. Refer to the troubleshooting guide to learn more about assay optimization.