ViewRNA ISH Tissue Colorimetric Assay Protocol

Visualizing RNA transcripts in tissue samples can provide insight into cellular processes maintained in healthy states or impacted in diseased conditions. Invitrogen ViewRNA uses branched DNA (bDNA) technology to enable single-molecule sensitivity in diverse sample types, such as FFPE and cryopreserved tissue. The colorimetric ViewRNA ISH Tissue Assay kits allow detection of up to 2 RNA transcripts at one time in a single sample using brightfield detection.

The assay kits are configured as ready-to-use 1-plex or 2-plex kits. For more flexibility, ViewRNA Core kit can be purchased with the individual Signal Development modules Type -1 (Fast Red), Type-4 (DAB) and Type-6 (Fast Blue). Fast Red and Fast Blue chromogens display a unique nature of being both colorimetric and fluorescent, allowing dual detection mode for result confirmation.

• Incubation times and temperatures should be strictly followed to ensure successful hybridization.
• All steps involving the use of RNase-free water should be performed using nuclease-free labware and disposable pipette tips to avoid RNase contamination.
• For the hybridization system, a humified tissue culture incubator is not recommended for this protocol.
• For all wash steps use 200 mL of indicated wash solution and use good washing technique. When performing washes, flick the slides to remove excess liquid carefully, do not completely dry the sample out.
• Prewarm Pre-Amplifier Mix, Amplifier Mix and Label Probe Diluent to 40°C On the day of the assay, before beginning the protocol.
• For 2-day protocol, prewarm the reagents on day 2. Due to the viscous nature of Diluent Reagents, it is suggested to not make aliquots, warm entire bottle to 40°C and remove liquid as needed.
• There is an optional overnight storage step.
• Chromogenic substrates are very sensitive and should be mixed right before use.

We recommend incorporating positive and negative controls in each assay to qualify and interpret your results.

Negative control examples

• Omit the target probe set
• Use a probe set designed to the sense strand of the target
• Use a probe set for a target that is not present in your tissue sample

Positive control examples

• Housekeeping genes: ACTB, GAPDH, or UBC (please refer to User Guide Page 39 for more information on control probe selection).

[1] Reagents have a minimum shelf life of 6 months from date of receipt when stored as recommended. See kit labels for expiration dates and the package insert for component quantities.

*ViewRNA Core kit QVT0400 comes with ViewRNA specific reagents that apply to all assays, regardless of type or plex. The core kit is to be supplemented with the individual modules that provide type-specific label and substrate reagents for signal development.
Alternatively, you can purchase the 1-plex or 2-plex kits that come with all reagents including the label probe and substrate. For individual catalog number information please refer to Appendix—Table 2.

1. 1x PBS: Prepare 3 L of 1X PBS (RNase-free)

2. Wash Buffer: Prepare 4 L of Wash Buffer according to the table below. Mix well with 3 L of water and adjust total volume by adding an additional 944 mL ddH₂O.

3. 1X Pretreatment Solution: Prepare 500 mL of 1X Pretreatment Solution according to the table below in a 1 L glass beaker.

4. Optional for overnight storage: Storage Buffer: Prepare 200 mL of Storage Buffer if using the optional stopping point according to the table below.

5. Thaw probe set(s) on ice. Mix, briefly centrifuge and place on ice until use.
6. Pre-warm entire bottle of Probe Set Diluent to 40°C.

Note: If performing entire assay in 1 day, also prewarm Amplifier and Label Probe Diluent to 40°C.

On the day of the assay:

1. Briefly centrifuge the following reagents, then place on ice until use.

2. Bring the following reagents to room temperature

Calibrate the temperature of the dry oven to 40°C using the ViewRNA Temperature Validation Kit. When using the dry oven and humidifying chamber such as StainTray slide holder, ensure that the appropriate water level is attained. StainTray holder can be replaced by an enclosed plastic chamber supplied with a wetted tissue paper and elevated platform to create a hybridization chamber.

Note: FFPE and cryopreserved tissues can be used in this protocol. Tissue preparation steps differ depending on the sample. Proceed to the appropriate section to prepare your tissue.

Bake the slides (optional)
Baking tissue samples at 60°C for 1 hour can promote tissue adhesion to sample slides. If you are making your own FFPE sample, this step may be required. If purchasing FFPE samples, please check with the provider to see if baking has already been conducted.

Deparaffinize the slides
Deparaffinize with xylene or Histo-Clear and rehydrate with Ethanol according to standard protocols.

Perform heat pretreatment

1. Cover the 1X Pretreatment Solution beaker tightly with aluminum foil and place on a hot plate. Heat to 90–95°C.
2. Place slides in a vertical slide rack and submerge into the heated 1X Pretreatment Solution using forceps. Cover the glass beaker with aluminum foil and incubate at 90–95°C for optimal time.
   Note: for guidelines, refer to user guide page 44 “Pretreatment optimization procedure”.
3. Remove slide rack with forceps and submerge slides into ddH₂O using a staining dish containing 200 mL of ddH₂O. Wash for 1 minute with frequent agitation.
4. Decant and repeat the wash with 200 mL of fresh ddH₂O.
5. Transfer the slide rack to a staining dish containing 1X PBS. Proceed to “Draw hydrophobic barrier” section.

Note: Do not let the tissue sections dry out from this point forward. After heat pretreatment, sections can be stored covered in 1X PBS at room temperature overnight.

Store slides (optional)
After heat pretreatment, slides can be stored overnight. Store slides in a clear staining dish containing 200 mL of Storage Buffer at room temperature for up to 24 hours. Cover the dish with a lid or sealing film to prevent evaporation.

Prepare compound-embedded tissue

1. Add tissue sections to pre-chilled 4% Formaldehyde (Image iT fixative solution) and fix overnight (16–19 hours) at 4°C.
2. Wash the slides with 200 ml 1X PBS for 1 minute. Proceed to “Draw hydrophobic barrier” section.

Store slides (optional)
After fixation, slides can be stored overnight. Store slides in a clear staining dish containing 200 mL of Storage Buffer at room temperature for up to 24 hours. Cover the dish with a lid or sealing film to prevent evaporation.

1. Use a Pap Pen to draw a hydrophobic barrier around the tissue section. Lightly trace the barrier 2 to 4 times. Cover tissue with 100–200 µL 1X PBS, enough to cover the sample completely without touching the hydrophobic barrier to ensure the tissue does not dry out.
2. Allow the barrier to dry at room temperature for 15–20 minutes.
3. If using the DAB Module, apply sufficient Peroxidase Quencher (instead of PBS) to cover the tissue while the barrier is drying to quench endogenous peroxidase activity. Incubate for 15–20 minutes at room temperature then rinse with ddH₂O.
4. Proceed to “Protease digestion and fixation” section.

Note: Ensure that 1X PBS is pre-warmed to 40°C before starting this procedure.

1. Prepare the working protease solution by diluting the Protease Solution 1:100 in prewarmed 1X PBS and briefly vortex.
2. Remove each slide from rack and flick to remove excess 1X PBS.
3. Place slides face up in the slide staining chamber and immediately add 400 µL of the working Protease Solution on the tissue section, making sure tissue section is covered.
4. Transfer slides to the dry oven and incubate at 40°C for optimal time. See user guide for tissue type and optimal incubation time guidance.
5. Decant the working protease solution from the slides, wash the slides 200 mL 1X PBS staining dish and gently agitate for 1 min. Repeat the wash 1 more time with fresh 1X PBS.
6. Transfer the slide rack to a staining dish containing 200 mL of fixation solution and fix for 5 minutes at room temperature under fume hood.
7. Wash the slides with 200 mL of fresh 1X PBS for 1 minute with frequent agitation.

Note: Ensure Probe Set Diluent is prewarmed to 40°C before starting this procedure. Prepare sufficient volume to add 400 µL per sample.

1. Prepare the working probe set solution by diluting the ViewRNA Probe Sets 1:40 in prewarmed Probe Set Diluent. Dilute 10 µL of each target probe set in prewarmed Probe Set Diluent to a reach total volume of 400 µL per slide. Vortex briefly to mix. See Table 3 in the Appendix for suggested dilutions for 1–2 plex assays.
   Note: For low-abundance targets, dilute target probe(s) 1:20 or 1:30.
2. Remove each slide from rack and flick to remove excess 1X PBS.
3. Add up to 400 µL of the pre-warmed Probe Set Diluent to the negative control and up to 400 µL of working probe set solution to each test sample.
4. Transfer the slides to the dry oven and incubate at 40°C for 2 hours.
5. Decant the working probe set solution from the slides and submerge slides into a slide rack containing Wash Buffer.
6. Wash the slides 3 times with fresh 200 mL Wash Buffer at room temperature for 2 minutes with constant agitation.
7. Proceed to “Store slides” if you plan to perform the assay over two days, otherwise proceed to “Amplify and detect signal”.

Store slides in a clear staining dish containing 200 mL of Storage Buffer at room temperature for up to 24 hours. Cover the dish with a lid or sealing film to prevent evaporation.

Note: Before beginning this step, make sure reagents for amplification and detection have been warmed to 40°C.

1. Remove from Storage Buffer. Transfer the rack to a clear staining dish containing Wash Buffer, and wash for 2 minutes with frequent agitation.
2. Decant Wash Buffer and wash again with 200 mL of fresh Wash Buffer for 2 minutes with frequent agitation. Repeat the wash 1 more time with fresh Wash Buffer.

1. Swirl the PreAmplifier Mix bottle briefly to mix the solution
2. Remove each slide and flick to remove Wash Buffer. Place slides face up on elevate platform and immediately add 400 µL of the PreAmplifier Mix on the tissue section, making sure tissue section is covered.
3. Transfer slides to the hybridization system and incubate at 40°C for 25 minutes.
4. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
5. Decant the PreAmplifier Mix from the slides and insert them into the slide rack in the Wash Buffer.
6. Wash the slides at room temperature for 2 minutes with constant agitation. Repeat this step with fresh Wash Buffer for a total of 3 washes.
7. Swirl the Amplifier Mix bottle to briefly mix the solution.
8. Remove each slide and flick to remove Wash Buffer. Place slides face up on elevate platform and immediately add 400 µL of the Amplifier Mix on the tissue section, making sure tissue section is covered.
9. Transfer slides to the hybridization system and incubate at 40°C for 15 minutes.
10. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
11. Decant the Amplifier Mix from the slides and insert them into the slide rack in the Wash Buffer.
12. Wash the slides at room temperature for 2 minutes with constant. Repeat this step with fresh Wash Buffer for a total of 3 washes.

Note: Ensure Label Probe Diluent is pre-warmed to 40°C before starting this procedure.

These steps can be followed for any of the colorimetric modules. If multiplexing, the optimal order is the DAB Module (Type 4 – HRP) followed by the Fast Blue Module (Type 6 – AP) followed by the Fast Red Module (Type 1 – AP). Perform the “Apply staining solution” steps immediately after the Label probe hybridization for each module. See Table 4 in the Appendix for steps for module combination.

1. Briefly centrifuge Label Probe before using.
2. Prepare the Working Label Probe Solution using the following table as a guide. Dilute Label Probe Type 1:1000 in prewarmed Label Probe Diluent and briefly vortex to mix.

3. Remove each slide and flick to remove the Wash Buffer. Place the slides face up on a flat, elevated platform and immediately add 400 µL of Working Label Probe Solution to each tissue section.
4. Transfer the slides to the hybridization system and incubate at 40°C for 15 minutes.
5. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
6. Decant the Working Label Probe Solution from the slides and insert them into the slide rack.
7. Wash the slides with 200 mL of fresh Wash Buffer at room temperature for 3 minutes with constant agitation. Repeat this step two more times for a total of 3 washes.
8. Proceed to “Apply staining solution”.

Important notes:

1. Chromogenic substrates are very sensitive and should be mixed right before use.
2. For 12 slides, prepare the staining solution in a 15 mL conical tube and vortex. Protect staining solutions from light by wrapping tubes in aluminum foil.
3. If multiplexing, the staining solutions steps should follow immediately after the label corresponding probe hybridization for each module. Follow the sequential order DAB → Fast Blue → Fast Red.

1. Prepare 500 µL of the DAB staining solution per slide. Refer to the table below for recommended dilution.

2. Remove each slide and flick it to remove the Wash Buffer. Add 400 µL of the appropriate staining solution. Incubate at room temperature for 2–15 minutes.
   Note: Monitoring the DAB color development by brightfield microscopy can help optimize development of the signal.
3. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
4. After incubation, decant the staining solution from the slides and insert them into the slide rack.
5. Wash the slides with 200 mL of fresh Wash Buffer at room temperature for 3 minutes with constant agitation. Repeat this step two more times for a total of 3 washes.
6. If multiplexing, repeat “Label Probe hybridization” for the next module. For 1-plex assays, proceed to “Counterstain and mount for detection”.

1. Prepare 500 µL of the Fast Blue staining solution per slide. Refer to the table below for recommended dilutions.

2. Remove each slide and flick it to remove the Wash Buffer. Add 400 µL of the appropriate staining solution. Incubate at room temperature for 30 minutes.
3. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
4. After incubation, decant the staining solution from the slides and insert them into the slide rack.
5. Wash the slides with 200 mL of fresh Wash Buffer at room temperature for 3 minutes with constant agitation. Repeat this step two more times for a total of 3 washes.
6. For 2-plex assays with Fast Blue/Fast Red, proceed to “Quench Label Probe Type 6 – AP”. For 1-plex assays or 2-plex assay with DAB/Fast Blue, proceed to ‘Counterstain and mount for detection”.

1. Remove each slide and flick it to remove the Wash Buffer. Tap the edge of the slides and wipe the backside with laboratory wipes, taking care to not completely dry out.
2. Place the slides face up on a flat, elevated platform, then immediately add 400 µL of AP Stop Solution.
3. Transfer to the moisture chamber, then incubate in the dark at room temperature for 30 minutes.
4. Insert an empty slide rack into a clear staining dish containing 200 mL of 1X PBS.
5. After incubation, decant the AP Stop Solution from the slides and insert them into the slide rack.
6. Wash the slides twice, each time in 200 mL of fresh 1X PBS at room temperature for 1 minute with frequent agitation.
7. Replace the 1X PBS with 200 mL of fresh Wash Buffer and rinse any residual PBS from the slides by moving the slide rack up and down for 1 minute.
8. Repeat steps starting from “Label Probe Hybridization” for the Fast Red module.

1. Remove each slide and flick it to remove the Wash Buffer. Place the slides face up on a flat, elevated platform and immediately add 400 µL of AP Enhancer Solution. Incubate at room temperature for 5 minutes while preparing staining solution.
2. Prepare 500 µL of Fast Red staining solution per slide. Refer to the table below for recommended dilutions.

3. Remove each slide and flick it to remove the Wash Buffer. Add 400 µL of the appropriate staining solution. Incubate at room temperature for 60 minutes.
4. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
5. After incubation, decant the staining solution from the slides and insert them into the slide rack.
6. Wash the slides with 200 mL of fresh Wash Buffer at room temperature for 3 minutes with constant agitation. Repeat this step two more times for a total of 3 washes.
7. Proceed to “Counterstain and mount for detection”.

1. For optimal results, follow the instructions provided with the reagent.
2. Proceed to mounting.

1. Perform DAPI counterstain according to standard protocols.

The ViewRNA Tissue Assay with DAB, Fast Blue and Fast Red substrates is compatible with most aqueous mountains. In addition to aqueous mountants, ViewRNA Tissue Assay DAB Module staining is compatible with xylene/ethanol dehydration and organic mountants. Avoid mountants containing alcohol when staining with the ViewRNA Tissue Assay Fast Blue Module and ViewRNA Tissue Assay Fast Red Module. Based on your detection mode, choose one of the following mountants.

1.1. For bright field or fluorescent imaging, mount under coverslips with an aqueous mountant:

• Histomount Mounting Solution
• ProLong Glass Antifade Mountant
• SlowFade Glass Soft-set Antifade Mountant

1.2. For 1-plex DAB colorimetric staining, organic moutnants can be used. (Note: Fast Blue is particularly soluble in xylene and not recommended for organic mountants)

• Epredia Cytoseal 60 Mountant

Note: For optimal results, follow the instructions provided with the mountant.

2. Analyze the tissue using a compatible imaging instrument such as EVOS Imaging Systems.

When mounted with a curing, hardening mountant, slides can be stored for years at 2–8°C.

Table 1. Definition of Colorimetric Probe set types.

Figure 1: Excitation and emission spectra for Fast Blue and Fast Red dye

Table 2.ViewRNA Tissue colorimetric kit and module options

*Core kit contains all reagents required to perform the experiment EXCEPT for colorimetric modules. Modules MUST be added in order to complete the kit.

Table 3. Examples of Target Probe Set dilutions.

Table 4. Label probe hybridization and staining solution steps for 1 and 2 plex modules.