May 07, 2025
Version 2
RNAscope Multiplex Fluorescent V2 Labeling of frog brains V.2
Version 1 is forked from Preparation and RNAscope-labeling of fresh mouse midbrain tissue
This protocol is a draft, published without a DOI.
- Billie Goolsby1,
- A. Zach Reddy1,
- Melody Joy Dailey1,
- Lauren A O'Connell2,
- William S Conrad2
- 1Stanford University;
- 2University of California San Diego
Protocol Citation: Billie Goolsby, A. Zach Reddy, Melody Joy Dailey, Lauren A O'Connell, William S Conrad 2025. RNAscope Multiplex Fluorescent V2 Labeling of frog brains. protocols.io https://protocols.io/view/rnascope-multiplex-fluorescent-v2-labeling-of-frog-gy29bxyh7Version created by Billie Goolsby
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2025
Last Modified: May 07, 2025
Protocol Integer ID: 217921
Keywords: rnascope, fresh tissue, sectioning
Funders Acknowledgements:
ASAP & MJFF
Grant ID: ASAP-020600
Gilliam Fellowship
Grant ID: GT15685
NIH R01
Grant ID: DP2HD102042
Disclaimer
While this guide has generally worked for one experimenter, this is not a replacement for doing your own research and preparing your own protocol. Each experimental design is different, and so is each experimenter. To have the best results possible, you should actively research, engage with the protocol, and consult with ACD.
Abstract
Below we describe preparing and labeling coronal sections of frog brains.
Attachments
Materials
Part A:
| A | B | |
| Material | Supplier and Catalog Number | |
| RNAseZap | Invitrogen (AM9780) | |
| Scissors | Fine Science Tools (14084-08) | |
| Forceps | Fine Science Tools (11242-40 ) |
Part B:
| A | B | |
| Material | Supplier and Catalog Number | |
| Cryostat | Leica (CM1860) | |
| Anti-roll plate | Leica (14047742497) | |
| Brushes | Electron Microscopy Sciences (66100-30) | |
| OCT Medium | Sakura (4583) | |
| Superfrost Plus Microscope Slides | Thermo Fisher Scientific (12-550-15) |
Part C & D:
| A | B | |
| Material | Supplier and Catalog Number | |
| Probes | RNAscope Target Probes (Catalog or Made-to-Order C1 to C3 Probes, not compatible with the RNAscope probes with T1-T3 designation). | |
| RNAscope Probe Diluent | Cat. No. 300041 | |
| RNAscope Multiplex Fluorescent Reagent Kit v2 (includes Pretreatment Kit, detection kit, wash buffer and TSA dilution buffer) | Cat. No. 323100 | |
| Wash Buffer | Advanced Cell Diagnostics (310091) | |
| HybEZ Oven | Advanced Cell Diagnostics (310010) | |
| Pretreatment Kit (Protease & hydrogen peroxide) | 322340 RNAscope™ Protease III & Protease IV Reagents 322330 RNAscope™ H202 & Protease Plus Reagents | |
| UltraPure DNase/RNase-Free Distilled Water | Invitrogen (10977049) | |
| Wash containers | Mortech Manufacturing (SP233) | |
| Safe Lock Centrifuge Tube, 1.5 mL | Eppendorf (0030123611) | |
| ImmEdge Hydrophobic Barrier PAP Pen | Vector Laboratories (H-4000) | |
| Prolong Gold Antimountant | Thermo Fisher (P36930) | |
| Coverslip | Corning (2980-225) | |
| Falcon 50 mL Conical Centrifuge Tubes | Fisher Scientific (352098) |
Before you start
Before you start
Please keep in mind that this protocol is designed for fresh frozen, adult frog brains. Tadpoles are much smaller, and can be dissected within their skull cap or have their brains dissected out. Think about how much the PFA has actually penetrate your brain tissue. Therefore, is your brain only lightly fixed, very fixed, or fresh? Make sure you are thinking thoughtfully about your experimental design before beginning. This protocol is not fail proof and no experiment is built the same, meaning that Billie is not responsible if this fails! Pilot and quality check your experiment before doing real samples.
Billie uses the fresh frozen (this method) for tadpoles that are dissected within their skull cap on and placed in PFA, and for adult brain that are snap frozen. However, you may have luck with other pretreatments, all of which are available in the ACD manual, and you can consult with the technical representatives for free, as well.
For other types of dissections, such as decalcification, can impact the integrity of DNA and RNA, which are necessary for in situ hybridization (ISH) techniques. While acid-based decalcification can be rapid, it may also damage nucleic acids, making them unsuitable for ISH. EDTA-based decalcification is generally preferred as it preserves DNA and RNA better, allowing for more reliable ISH results.
A. Frog brain extraction
A. Frog brain extraction
Spray experimenter’s gloves and all tools with RNAseZap before starting and in between brain extractions. Wipe RNAseZap away with RNAse free water or ethanol, as RNAseZap has SDS. Prepare an eppendorf tube for embedding by labeling it, cutting off the conical tip, closing the lid, and fill with OCT. Place on dry ice.
Alternative: for PFA fixation, prepare 4% PFA and put on ice.
Prepare an eppendorf tube for embedding by labeling it, cutting off the conical tip, closing the lid, and fill with OCT. Place on dry ice.
Alternative: put ~500 uL of 4% PFA in an eppendorf tube, and put on regular ice.
After following APLAC procedures for anesthesia, decapitate frog and extract brain from skull. Gently cut skull casing along the side so that the skull capsule containing the brain is exposed, but you did not cut the brain itself. Using two forceps, pull part the skull casing exposing the brain. Gently remove brain with forceps (careful - optic nerves, may still be attached at the bottom and may need to be severed with microscissors or fine forceps).
Image courtesy of Biorender.
Image courtesy of Biorender.
Immediately snap freeze brain by placing the tube on dry ice. Make sure the brain freezes correctly.
Alternative: Put brain/tissue in PFA eppendorf tube. Let fix overnight. After 24 hrs, move brain to a new tube, replace 4% PFA with 1X rnase free PBS. After 24 hrs, move brain to a new tube, fill with 30% sucrose. After 24 hrs, remove the brain and embed in OCT as described above. If you do a PFA fixation, and you are not doing tadpoles that still have their heads, you are doing a new protocol that requires your own development. In that case, please refer to the ACD manual.
Frozen brains can be moved to either an RNAse-free cryostat at −18°C or stored at −80°C.
B. Sectioning
B. Sectioning
Set temperature of cryostat objective and chamber to −18°C.
Clean interior of cryostat with 100% ethanol.
https://www.protocols.io/view/rnascope-multiplex-fluorescent-v2-labeling-of-frog-gyxnbxxmfBefore sectioning, spray experimenter’s gloves and tools with RNAseZap. Tools that directly touch sections (e.g. brushes, anti-roll plate) should be air dried completely before being placed in cryostat. Do not rub your hands as you will cause static electricity that can affect your sections.
Place culture tube with brain in chamber and let it equilibrate to chamber temperature for about 10 minutes.
For coronal sections, mount caudal end of brain to chuck using OCT mounting medium. Mount brain as perpendicular to the horizontal plane of the chuck as possible. Equilibrate to chamber temperature again for about 10 minutes.
Place chuck with brain on objective and adjust orientation to make brain perpendicular to blade. As you begin cutting, adjust brain position so brain landmarks (optic tectum) are as symmetrical as possible. Ensure anti-roll plate is positioned correctly before reaching area of interest.
Cut coronal brain sections serially at 15-18 μm on a cryostat throughout the rostro-caudal extent of the region of interest (ROI).
Directly mount sections onto Superfrost glass slides. Mount within the white cross hatches of the slide only.
Air-dry at room temperature (RT) for 10-20 minutes, then store at -80°C in a slide box within an airtight container (e.g. Ziplock bag).
D. RNAscope assay Day 1
D. RNAscope assay Day 1
Turn on HybEZ™ Oven (ACD) and set to 40°C.
Warm probes in heat-bath for 10 minutes at 40°C, then cool to RT.
Make probe mixture in an RNAse free Eppendorf tube by pipetting (with filter pipette tips) 50 parts C1 probe or diluent, 1 part C2 probe (optional) and 1 part C3 probe (optional). Prepare about 150 µL per slide, assuming each slide contains ~20-25 brain sections.
Lay slides flat, tissue side up on a clean surface. Fix slides in RNAse-free PBS containing 4% paraformaldehyde at RT for 60 minutes. Keep slides covered to minimize fumes.
Take out ACD hydrogen peroxide and protease kit to reach room temperature.
Dehydrate slides flat in serial ethanol (EtOH) washes: 2 minutes in 50% EtOH, 2 minutes in 70% EtOH, 2 minutes in 100% EtOH twice. Make EtOH dilutions with DI or milliq water in sterile falcon tubes.
Air dry slides flat for 5 minutes.
Use hydrophobic pen to draw a barrier around sections. Air dry for 5 minutes.
Put slides into slide holder. Have the white component of the slide face outward from the center of the slide holder so that you can access your tissue on your slide.
Incubate slides flat with hydrogen peroxide at RT for 15 minutes. Keep slides covered to prevent dust contamination.
Note: If you run out of hydrogen peroxide, you can use 3% H2O2 made in lab. Take 1 mL of 30% H2O2, which is in lab, and fill to 10 mL with DI H2O.
Rinse slides in RNAse-free DI (not milliq) water at RT, twice. Do flat.
Incubate slides flat with protease IV at RT for 30 min. Keep slides covered to prevent dust contamination.
Rinse slides in RNAse-free DI (not milliq) water at RT, twice. Do flat.
Decant excess liquid from slides by gently tapping the slide edge on a paper towel. Pipette enough probe mixture to cover sections (~150 uL).
Incubate for 2 hours in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Fill wash tray with 5X SSC, and place slide holder inside. Make sure all slides are covered and that the wash tray is covered a lid. Leave overnight at RT.
Prepare 200 mL of 5X SSC by diluting 50 mL of 20X SSC with 150 mL distilled water. 20X SSC is available online.
This step is optional and can be skipped. If you do skip it, you cannot stop the protocol until it is complete.
D. RNAscope assay Day 2
D. RNAscope assay Day 2
Prepare fluorophore dilutions.
There are 520, 570, and 650 fluorophores. This is approximately equivalent to 488, 555, and 647 in IHC. These fluorophores are specific for RNAscope, do not use them for any other experiment. There are ~10 uL aliquots in the -80. Take one aliquot out and keep it in your 4C until you have finished using it. It will be solid at 4C, as the fluorophores are reconstituted in DMSO.
Do not mix the fluorophores. Assign one fluorophore to one gene channel (C1,C2,C3). Keep track and write it down.
We recommend starting with a dilution of 1:1500 for TSA Vivid 520, 570, and 650 and adjusting the dilution based on signal intensity. Optimal fluorophore dilutions may vary based on sample, target expression levels, and imaging system. Dilute in TSA buffer.
Example: For C1, I will use the 520 fluorophore. I take out the fluorophore from my 4, and let it reach room temp so that the DMSO turns to liquid. I will add 1.26 uL of the 520 fluorophore to 1,800 uL of TSA buffer. I will use it at step 42. And so on and so forth....
If you stored your slides in SSC, wash in 1X wash buffer for 2 minutes, once. If you skipped the SSC step, skip this step also.
Decant excess liquid from slides and add enough AMP1 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Decant excess liquid from slides and add enough AMP2 to cover sections. Incubate for 30 minutes in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Decant excess liquid from slides and add enough AMP3 to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Decant excess liquid from slides and add enough HRP-C1 to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Add diluted fluorophore for labeling the C1 probe and incubate for 30 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Add HRP blocker to slides and incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Decant excess liquid from slides and add enough HRP-C2 to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1X wash buffer for 2 minutes, twice.
Add diluted fluorophore for labeling the C2 probe and incubate for 30 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Add HRP blocker to slides and incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Decant excess liquid from slides and add enough HRP-C3 to cover sections. Incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Add diluted fluorophore for labeling the C3 probe and incubate for 30 minutes in the HybEZ™ Oven at 40°C.
Add HRP blocker to slides and incubate for 15 minutes in the HybEZ™ Oven at 40°C.
Wash in 1x wash buffer for 2 minutes, twice.
Counterstain and mounting
Counterstain and mounting
Before you start: Do this procedure with no more than five slides at a time.
Decant excess liquid from slides and add enough DAPI to cover each section and incubate for 30 seconds at RT.
Remove DAPI by tapping or flicking the slides. Immediately place 1-2 drops of ProLong Gold Antifade mountant on each slide.
Carefully place coverslip on the tissue section. Avoid air bubbles.
Dry the slides overnight in the dark RT.
Seal edges of slides with clear nail polish.
Store slides in the dark at 2-8 °C.