RNA CISH Signal Detection: A Technique to Detect Chromogenic Signals During RNA In Situ Hybridization in Intact Tissue Specimens

Protocol

1. Preparation of Counterstaining Reagents

1. Prepare 50% hematoxylin.
   1. In a fume hood, add 100 mL of Gill’s hematoxylin I (see the Table of Materials) to 100 mL of distilled water in a staining dish.
      NOTE: The 50% hematoxylin staining solution can be reused for up to 1 week.
2. Prepare 0.02% (w/v) ammonia water (bluing reagent).
3. In the fume hood, add 1.43 mL of 1 N ammonium hydroxide to 250 mL of distilled water in a graduated cylinder or another container. Seal the cylinder with paraffin film. Mix its contents well for 3x–5x.
   NOTE: For assay quantitation, it is critical to use ammonium hydroxide. The reagents may be prepared ahead of time. Ensure all containers remain covered.

2. Signal Detection with 3,3'-diaminobenzidine

CAUTION: Diaminobenzidine (DAB) is toxic. Follow appropriate precautions and safety guidelines when disposing of and handling this chemical.

1. Mix equal volumes of DAB-A and DAB-B (see the Table of Materials) in an appropriately sized tube by dispensing the same number of drops of each solution. Make ~120 µL of DAB substrate per section (~2 drops of each reagent/total of 4). Mix it well for 3x–5x.
2. Take each slide, one at a time, from the slide rack and tap and/or flick to remove the excess liquid before placing it in the slide rack.
3. Pipette ~120 μL of DAB onto each tissue section. Ensure the sections are covered and incubate for 10 min at room temperature.
4. Dispose the remaining DAB according to local regulation and insert the slide into a slide rack submerged in a staining dish filled with tap water.

3. Counterstaining

1. Move the slide rack to a staining dish containing 50% hematoxylin staining solution let it rest for 30 s at room temperature. Note that the slides will become purple.
2. Immediately transfer the slide rack back to a staining dish containing tap water and wash the slides 3x–5x by moving the rack up and down.
3. Keep repeating the washing step with fresh tap water until the slides are clear while sections remain purple.
4. Replace the tap water in the staining dish with 0.02% ammonia water. Move the rack up and down 2x–3x. Note that the tissue section should turn blue.
5. Replace the ammonia water with tap water. Wash the slides 3x–5x.

4. Dehydration

1. Move the slide rack to a staining dish containing 70% ethanol in the fume hood and let it rest for 2 min with occasional agitation.
2. Move the slide rack to a first staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.
3. Move the slide rack to a second staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.
4. Move the slide rack to a staining dish containing xylene and let it rest for 5 min with occasional agitation.

5. Slide Mounting

1. Remove the slides from the slide rack and lay them flat, with the sections facing up in the fume hood.
2. Mount one slide at a time by adding 1 drop of a xylene-based mounting medium to each slide and carefully placing a 24 mm x 50 mm coverslip over the section. Avoid trapping any air bubbles.
3. Air-dry the slides for ≥5 min.

6. Sample Evaluation

1. Examine the tissue sections under a standard brightfield microscope at 20x–40x magnification.

Materials

Name	Company	Catalog Number	Comments
Hematoxylin solution, Gill No. 1	Merck	GHS132
RNAscope 2.5 HD Detection Reagents-BROWN	Advanced Cell Diagnostics Inc.	322310	This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B
RNAscope Probe- HPV16/18	Advanced Cell Diagnostics Inc.	311121
HybEZ slide rack	Advanced Cell Diagnostics Inc.	300104
ImmEdge Hydrophobic Barrier Pen	Advanced Cell Diagnostics Inc.	310018