ViewRNA Tissue Fluorescence Assay Protocol

• 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 Diluent Reagents to 40°C before beginning the protocol. 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.

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 13 for more information on control probe selection)

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 slide 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.

FFPE tissue preparation

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

1. Deparaffinize with xylene 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 29 “ Pretreatment optimization procedure”).
3. Remove slide rack with forceps and submerge slides into dd H₂O using a staining dish containing 200 mL of dd H₂O. Wash for 1 minute with frequent agitation.
4. Decant and repeat the wash with 200 mL of fresh dd H₂O.
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)

1. 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.

Cryopreserved tissue preparation

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)

1. 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.

Draw hydrophobic barrier

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. Proceed to “Protease digestion and fixation” section.

Protease digestion and fixation

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 and add 400 µL per slide. See Table 3 in the Appendix for suggested dilutions for 1–4 plex assays.
   Note: For low-abundance targets, dilute target probe(s) 1:20 or 1:30. Remove each slide from rack and flick to remove excess 1X PBS.
2. 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.
3. Transfer the slides to the dry oven and incubate at 40°C for 2 hours.
4. Decant the working probe set solution from the slides and submerge slides into a slide rack containing Wash Buffer.
5. Wash the slides 3 times with fresh 200 mL Wash Buffer at room temperature for 2 minutes with constant agitation.
6. Proceed to “Store slides” if you plan to perform the assay over two days, otherwise proceed to “Amplify and detect signal”.

Store slides (optional)

1. 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.

Wash stored slides (skip for one-day protocol)

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.

Perform signal amplification

Note: Ensure that Amplifier Diluent QF is pre-warmed to 40°C before starting this procedure.

1. Prepare working Pre-Amplifier Mix solution by mixing and then diluting 1:25 in pre-warmed Amplifier Diluent QF.
2. Remove each slide and flick to remove Wash Buffer. Place slides face up on slide staining chamber and immediately add 400 µL of the working Pre-Amplifier Mix on the tissue section, making sure tissue section is covered.
3. Transfer the slides to the hybridization system and incubate at 40°C for 30 minutes.
4. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
5. Decant the Pre-Amplifier 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. Prepare the working Amplifier Mix solution by briefly swirling and diluting 1:25 in pre-warmed Amplifier Diluent QF.
8. Remove each slide and flick to remove Wash Buffer. Place slides face up on slide staining chamber and immediately add up to 400 µL of the Amplifier Mix on the tissue section, making sure tissue section is covered.
9. Transfer the slides to the hybridization system and incubate at 40°C for 30 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 agitation. Repeat this step with fresh Wash Buffer for a total of 3 washes.

Perform label probe hybridization

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

1. Prepare working Label Probe Mix solution by briefly swirling and diluting label probes 1:25 in prewarmed Label probe Diluent QF. Dilute 16 µL of each label probe in prewarmed Label Probe Diluent to reach a total volume of 400 µL per slide. Vortex briefly to mix. See Table 4 in the Appendix for suggested dilutions for 1–4 plex assays.
2. Vortex briefly to mix and protect from light.
3. Remove each slide and flick to remove Wash Buffer. Place slides face up on slide staining chamber and immediately add 400 µL of the pre-warmed working Label Probe Mix on the tissue section, making sure tissue section is covered.
4. Transfer slides to the hybridization system and incubate at 40°C for 30 minutes.
5. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer.
6. Decant the Label Probe Mix from the slides and insert them into the slide rack in the Wash Buffer.
7. Wash the slides at room temperature for 2 minutes with constant agitation. Repeat this step with fresh Wash Buffer.
8. Perform a final wash with Wash Buffer for 10 minutes with frequent agitation. Do not wash longer than 30 minutes.
9. Proceed to “Counterstain and mount for detection”.

(Optional): Reduce tissue autofluorescence

1. Remove each slide from the Wash Buffer, then flick to remove excess Wash Buffer.
2. Wash the slides three times with fresh 1X PBS for 3 minutes at room temperature with frequent agitation.
3. Prepare the ReadyProbes Tissue Autofluorescence Quenching Kit by combining equal volumes of Components A, B, and C.
4. Add 400 µL of the prepared ReadyProbes solution to each sample, then incubate for 5 minutes at room temperature.
5. Wash the slides three times with fresh 1X PBS for 3 minutes at room temperature with frequent agitation.
6. Proceed to “Counterstain and mount for detection”

Counterstain and mount for detection

Note: For the ViewRNA Tissue Fluorescence Assay, mounting media with antifade are highly recommended.

1. Counterstain the tissue with DAPI using standard protocols.
2. Mount the coverslips using a mountant with antifade properties. ProLong Glass reagent is recommended for hard-set mounting and long-term archiving of tissue samples. SlowFade Glass reagent is recommended for soft-set mounting if coverslip removal is required.
   Note: For optimal results, follow the instructions provided with the mounting reagent.
3. Analyze the tissue using a compatible imaging system such as inverted fluorescent microscope or HCS platform.

Table 1. Definition of Fluorescence Probe set types.
Spectral properties of these fluorophores can be found in our online fluorophore selection guides.

Table 2. ViewRNA Tissue fluorescence kit and module options.

Table 3. Examples of Target Probe set dilutions.

Table 4. Suggested Label Probe dilutions.