RNAscope® is a cutting-edge technique that enables the visualization of individual RNA molecules within individual cells and tissue. This assay, based on Advanced Cell Diagnostics patented technology, allows researchers to examine gene expression patterns and identify specific RNA targets in fixed cell and tissue samples. With its high sensitivity and specificity, RNAscope® holds immense potential in various fields, including cancer research, cardiology, and neuroscience. At Anilocus, we utilize this technique for multiple therapeutic areas and our CRO services. By providing valuable insights into gene expression at the single-cell level, RNAscope® represents a powerful tool for understanding cellular processes and identifying potential biomarkers or therapeutic targets. Check out our comprehensive RNAscope® protocol for fixed frozen rodent brain slide-mounted sections.

DAY 1 (Duration: ~1.5hrs)

OCT Wash & Post-Fixation (Duration: ~60 mins)

  1. Make sure incubator is on and set to 60°C.
  2. WASH slides in 1X PBS for 5 minutes to remove the OCT.
  3. BAKE slides for 30 mins at 60°C in the incubator.
  4. POST-FIX slides in prechilled 10% NBF or 4% PFA for 15 minutes at 4°C.

Tissue Dehydration (Duration: ~25 mins)

  1. Fill coplin jars with the following EtOH: 50%, 70%, and 100%
  2. Drop slides in 50% EtOH for 5 minutes at room temperature (RT).
  3. Drop slides in 70% EtOH for 5 minutes at RT.
  4. Drop slides in 100% EtOH for 5 minutes at RT.
  5. 15 Remove slides from 100% EtOH and let them air dry OVERNIGHT.

DAY 2 (Duration: ~5hrs)

RNAscope® Hydrogen Peroxide (Duration: ~45 mins)

  1. Turn on the Ez-Bake Oven; confirm the tempature is set to 40°C.
  2. Place a layer of blotting paper cut from roll (Thin Blot Paper #1620118) or using the precut paper to cover;
  3. Dampen the layer of absorbent paper with dH2O (but do not soak it)
  4. Place the tray into the oven and warm the tray for 30 MINS at 40°C (always keep the tray in the oven when not in use)
  5. Prepare 265mL of RNAscope® 1X Target Retrieval Reagents by adding 238mL distilled water to 27mL 10X target retrieval reagents.
  6. Draw hydrophobic barrier around tissue (not yet if using the historack).
  7. Add ~5 drops of RNAscope® Hydrogen Peroxide to each section to cover the sections.
    • INCUBATE: 10 min @ RT
  8. WASH: place slides in wash tray filled with dH2O; wash 2 x 2 mins each time.
    • Rock those slides around in the tray carefully washing them. Flick off the excess liquid.

Target Retrieval (Duration: ~70 mins)

  1. Fill the steamer with water and start it up at the maximum time.
  2. Add your two coplin jars that contain 1X Target Retrieval Reagent and dH2O. 
  3. Keep two coplin jars outside the steamer filled with dH2O and the other with 100% EtOH.
  4. After 30 minutes take the temperature from the dH2O coplin jar and make sure it reaches at least 99°C.
  5. Refill the steamer (pour water through the center part).
  6. Add slides to the HOT dH2O coplin jar for 10 seconds to acclimate the slides.
  7. Switch the slides to the HOT Target Retrieval jar and cover the steamer for 15 minutes (for mild conditions).
  8. Remove slides from Target Retrieval jar and add them to jar with dH2O at RT.
  9. Transfer from dH2O to 100% EtOH for 3 minutes.
  10. Dry slides in 60°C incubator for 5 minutes.
  11. *REAPPLY BARRIER* it was washed off after 100% EtOH!
  12. Allow barrier to dry at least 15 minutes at RT.

Protease III (Duration: ~45 mins)

  1. Load slides into the holder frosted side away from center.
  2. Add ~5 drops of Protease III to each section.
    • INCUBATE: 30 min @ 40°C
  3. WASH: place slides in wash tray; wash 2 x 2 mins each time.

Probe Hybridization (Duration: ~2.0 hours)

  1. Add 4-6 drops of C1 probe to each slide covering tissue completely.
  2. C1: 50µl → uncap the ready to use probe but do not dilute it (use at least 25uL per brain section)
  3. C2: 1µl of 50x C2 probe (combine with C1) C3: 1µl of 50x C3 probe (combine with C1+C2)
    • INCUBATE: 2 hours at 40°C.
  4. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  5. Store slides in 5x SSC overnight at RT.
  6. Prepare SSC: 125mL 20x SSC + 375mL dH2O.

DAY 3 (~6hrs)

Hybridization Amplification (Duration: ~1.5 hours)

  1. Turn on the Ez-Bake Oven; confirm the temperature is set to 40°C.
  2. WASH: place slides in wash tray; wash 2 x 2 mins each time. Rock those slides around.
  3. HYBRIDIZE AMP 1: Add 4-6 drops of RNAscope® Multiplex FL v2 AMP 1;
    • INCUBATE: 30 min @ 40°C then WASH: place slides in wash tray; wash 2 x 2 mins each time.
  4. HYBRIDIZE AMP 2: Add 4-6 drops of RNAscope® Multiplex FL v2 AMP 2;
    • INCUBATE: 30 min @ 40°C then WASH: place slides in wash tray; wash 2 x 2 mins each time.
  5. HYBRIDIZE AMP 3: Add 4-6 drops of RNAscope® Multiplex FL v2 AMP 3;
    • INCUBATE: 30 min @ 40°C then WASH: place slides in wash tray; wash 2 x 2 mins each time.

Signal Development: HRP-C1 Signal (Duration: ~1.25 hrs)

  1. 44 Add 4-6 drops RNAscope® Multiplex FL v2 HRP-C1
    • INCUBATE: 15 mins at 40°C.
  2. While slides are incubating in HRP-C1 preapre the Opal 520 dye and use a concentration of 1:1000. Also take out your DAPI Hardset from freezer! Other colors: 520 (GFP), 570 (Cy3), 620, 690 (Cy5).
  3. Ex: 1000µL TSA buffer + 1µL of Opal 520 dye.
  4. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  5. Add Opal 520 dye to each section cover the section completely.
    • INCUBATE: 30 mins at 40°C.
  6. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  7. Add 4-6 drops RNAscope® Multiplex FL v2 HRP-Blocker
    • INCUBATE: 15 mins at 40°C.
  8. WASH: place slides in wash tray; wash 2 x 2 mins each time.

Signal Development: HRP-C2 Signal (Duration: ~1.25 hrs)

  1. Add 4-6 drops RNAscope® Multiplex FL v2 HRP-C2
    • INCUBATE: 15 mins at 40°C.
  2. While slides are incubating in HRP-C2 prepare the Opal 570 dye and use a concentration of 1:1000.
    • (Ex: 1000µL TSA buffer + 1µL of Opal 570 dye.)
  3. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  4. Add Opal 570 dye to each section cover the section completely.
    • INCUBATE: 30 mins at 40°C.
  5. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  6. Add 4-6 drops RNAscope® Multiplex FL v2 HRP-Blocker
    • INCUBATE: 15 mins at 40°C.
  7. WASH: place slides in wash tray; wash 2 x 2 mins each time.

Signal Development: HRP-C3 Signal (Duration: ~1.25 hrs)

  1. Add 4-6 drops RNAscope® Multiplex FL v2 HRP-C3
    • INCUBATE: 15 mins at 40°C.
  2. While slides are incubating in HRP-C3 prepare the Opal 620 dye and use a concentration of 1:1000.
  3. (Ex: 1000µL TSA buffer + 1µL of Opal 620 dye.)
  4. 60 WASH: place slides in wash tray; wash 2 x 2 mins each time.
  5. Add Opal 620 dye to each section cover the section completely.
    • INCUBATE: 30 mins at 40°C.
  6. WASH: place slides in wash tray; wash 2 x 2 mins each time.
  7. Add 4-6 drops RNAscope® Multiplex FL v2 HRP-Blocker
    • INCUBATE: 15 mins at 40°C.
  8. WASH: place slides in wash tray; wash 2 x 2 mins each time.

Counterstain & Mount Slides (Duration: ~45 mins)

  1. Add DAPI in PBS to slides (1:1000) and incubate in black slide chamber for 15 mins.
  2. Coverslip the slides using Fluoromount-G™ Mounting Medium.
  3. DRY SLIDES for at least 30 minutes to OVERNIGHT in the dark at 4°C.

Check out our comprehensive protocols for in situ hybridization, immunohistochemistry, and more!