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

All procedures involving human participants have been performed in compliance with the institutional, national and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of the Materials

1. Preparation of 1x target retrieval reagent
   1. In a large beaker, add 70 mL of 10x target retrieval reagent (see the Table of Materials) to 630 mL of distilled water.
   2. Place the beaker on a heating plate with a magnetic stirrer. Cover it with aluminum foil.
   3. Boil its contents at 100 °C for 10–15 min.
      NOTE: Do not let it boil for more than 30 min.

2. Tissue Pretreatment

NOTE: These steps follow the “standard” pretreatment recommendation according to the manufacturer’s instructions for head and neck samples. The timing of sections 2.1 and 2.2 may need to be adjusted depending on the manipulated tissue.

1. Blockade of peroxidase activity
   1. Add 4–6 drops of hydrogen peroxide (see the Table of Materials) to each slide and incubate them for 10 min at room temperature.
   2. Wash the slides 2x for 2 min in distilled water at room temperature.
2. Breakage of RNA/tissue bounds
   1. With a claw, remove the aluminum foil from the boiling 1x target retrieval reagent (TTR1x) placed on a heating plate and stop stirring. Immerse the slide rack slowly and very carefully for 15 min. Cover the beaker again with aluminum foil.
      NOTE: Simmering must persist during this step.
      CAUTION: Use the claw to manipulate the aluminum foil and the slide rack to avoid burn injuries. Make sure to wear proper personal protective equipment, such as gloves and a lab coat.
   2. With the claw, immediately transfer the hot slide rack to a distilled water bath and wash it for 2 min.
      NOTE: Make sure the samples do not cool down in the TTR1x.
   3. Wash the slides in fresh 100% ethanol for 2 min.
   4. Let the slides dry at room temperature for 2 min.
3. Barrier creation
   1. With a hydrophobic barrier pen (see the Table of Materials), draw a barrier around the sample. Let it dry for at least 5 min.
      NOTE: Allow the barrier to really dry out. If it wears out during the procedure, do not hesitate to draw it again. Avoid touching the tissue with the pen. The protocol can be paused overnight here.
4. Protease digestion
   1. Place the slides on the slide rack and add ~4 drops of Protease Plus per sample (see the Table of Materials).
   2. Cover the humidity control tray with a lid and insert it into the hybridization oven for 30 min at 40 °C.
      NOTE: To prevent evaporation, make sure the turn knob is completely turned to the lock position.
   3. Remove the tray from the oven and remove the slide rack.
   4. One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with distilled water.
   5. Wash the slides 2x for 2 min in distilled water at room temperature. Agitate constantly.