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

Pretreatment of formalin-fixed paraffin-embedded or FFPE tissue sections facilitates the exposure of nucleic acids and enables the binding of labeled probes to the target sequence during subsequent in situ hybridization. In addition, pretreatment helps maintain the integrity of the nucleic acids within the cells and overall tissue morphology.

To begin, take a glass slide carrying a deparaffinized and rehydrated FFPE tissue section of interest. Treat the tissue with an appropriate concentration of hydrogen peroxide and incubate.

Hydrogen peroxide irreversibly inactivates the endogenous peroxidase, which is physiologically present in the cells. This blocking process eliminates the possibility of non-specific background signals during subsequent hybridization steps.

Next, immerse the tissue section in a heated bath containing an appropriate antigen retrieval buffer. Incubate for the desired duration. High temperature cleaves fixative-induced chemical crosslinks in proteins and nucleic acids, exposing the unmasked epitopes to probes during subsequent labeling.

Finally, treat the specimen with a suitable protease solution. Incubate under a humidity-controlled environment. Proteases increase the accessibility of RNA, facilitating the specific probes to reach their target sequence in the following hybridization steps.

Wash the section with water to remove any residual proteases. Use the pretreated tissue section 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.