Probe Hybridization and Signal Amplification in RNA In Situ Hybridization: A Technique for Detecting Specific RNA Sequences in Tissue Sections

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. Running the Assay

NOTE: Do not let sections dry out between the incubation steps.

1. Hybridization of the HPV probe
   NOTE: Ensure the probes are prewarmed to dissolve any precipitation prior to use. For this step, instead of HPV probe, peptidyl-prolyl isomerase B (PPIB) for positive control or dihydrodipicolinate reductase (DAPB) for negative control (see the Table of Materials) may also be used.
   1. Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of HPV probe to entirely cover each section.
   2. Cover the tray with a lid and insert it into the oven for 2 h 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 1x wash buffer.
   5. Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.
2. Hybridization of AMP1, AMP2, AMP3, and AMP4
   NOTE: These steps include hybridization in the hybridization oven and AMP1–AMP4 from the purchased kit (see the Table of Materials).
   1. Tap and/or flick to remove any excess liquid from the slides and place them in the slide rack. Add ~4 drops of AMP1 to entirely cover each section.
   2. Cover the tray with a lid and insert it into the oven for 30 min at 40 °C.
   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 1x wash buffer.
   5. Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.
   6. Repeat steps 1.2.1–1.2.5, but use ~4 drops of AMP2 instead of AMP1 and incubate for 15 min at 40 °C. Wash the slides 2x for 2 min, both times in fresh wash buffer.
   7. Repeat steps 1.2.1–1.2.5, but use ~4 drops of AMP3 instead of AMP1 and incubate for 30 min at 40 °C. Wash the slides 2x for 2 min, both times in fresh wash buffer.
   8. Repeat steps 1.2.1–1.2.5, but use ~4 drops of AMP4 instead of AMP1 and incubate for 15 min at 40 °C. Wash the slides 2x for 2 min, both times in fresh wash buffer.
3. Hybridization of AMP5 and AMP6
   ​NOTE: These steps do not include hybridization in the oven but hybridization at room temperature. AMP5 and AMP6 come from the purchased kit (see the Table of Materials).
   1. Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of AMP5 to entirely cover each section.
   2. Cover the tray with a lid and incubate for 30 min at room temperature.
   3. One slide at a time, quickly remove any excess liquid and place it in a slide rack submerged in a staining dish filled with 1x wash buffer.
   4. Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.
   5. Repeat steps 1.3.1–1.3.4, but use ~4 drops of AMP6 instead of AMP5 and incubate for 15 min at room temperature. Wash the slides 2x for 2 min, both times in fresh wash buffer.