FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In situ Hybridization

To block peroxidase activity on slides with tissue sections previously deparaffinized and placed in a slide rack, add 4 to 6 drops, just enough to cover the sample, of hydrogen peroxide to each slide and incubate them for 10 minutes at room temperature. Wash the slides two times for 2 minutes in distilled water at room temperature. To break RNA tissue bounds and the tissue sections, first, remove the aluminum foil from the boiling TRR1x using a claw and stop stirring.

Immerse the slide rack slowly and very carefully. Cover the beaker again with the aluminum foil and incubate for 15 minutes. Use the claw to immediately transfer the hot slide rack to a distilled water bath and wash it for 2 minutes. Wash the slides then in fresh 100% ethanol for 2 minutes and let them dry at room temperature for 2 minutes.

Next, use a hydrophobic barrier pen to draw a barrier around each sample and let it dry out for at least 5 minutes. For protease digestion, place the slides in the humidity control tray and add approximately 4 drops of Protease Plus per sample. Cover the tray with a lid and insert it into the hybridization oven for 30 minutes at 40 degrees Celsius.

Remove the tray from the oven and remove the slide rack. Working one slide at a time, quickly remove any excess liquid and place the slide in the slide rack submerged in a staining dish filled with distilled water. Wash the slides two times for 2 minutes in distilled water at room temperature with constant agitation.