Nov 18, 2025
Fresh Frozen RNAScope Protocol
- Lisa Kim1
- 1Northwestern University
Protocol Citation: Lisa Kim 2025. Fresh Frozen RNAScope Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqp83kvk5/v1
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: November 18, 2025
Last Modified: November 19, 2025
Protocol Integer ID: 232866
Keywords: ASAPCRN, acd bio rnascope multiplex fluorescent v2, frozen mouse brain tissue, mouse brain, cryosectioning method
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-025188
Abstract
A refined protocol for ACD Bio RNAscope Multiplex Fluorescent v2 in situ hybridization (ISH), tailored for reliable use with fresh-frozen mouse brain tissue. This guide provides a step-by-step workflow, including essential sample preparation and cryosectioning methods. The protocol is validated specifically for the mouse brain, but may be applied to other central nervous system areas.
Guidelines
1. Under-fixation of tissue specimens will result in protease over-digestion, which leads to loss of RNA and poor tissue morphology. Over-fixed tissue specimen will result in protease under-digestion, which leads to poor probe accessibility and low signal and signal/background ratio while maintaining excellent tissue morphology.
2. Fixed frozen at 7-15um sections, tissue pretreatment using Target Retrieval and Protease III
3. Depends on the probe, but I’ve noticed signal fades away 2 weeks after perfusion. Try to do all steps as soon as possible after perfusion.
Materials
Materials
- 1x PBS (using autoclaved H2O)
- DDW
- OCT Compound or FSC 22 Clear (Leica, #3801480)
- 2-methylbutane
- methanol
- SuperFrost Plus slides (Fisherbrand, #1255015)
- Microscope Cover Glass 24x60mm No. 1.5 (Fisherbrand, #12541037)
- 4% PFA in 1x PBS
- Fresh 50%, 70%, 100% EtOH
- ImmEdge Hydrophobic Barrier Pen
- Target Probes in C1, C2 and/or C3
- Positive and/or Negative Control Probes
- Probe Diluent (Cat #300041) if using C2 or C3 without C1
- RNAScope Multiplex Fluorescent Reagent Kit v2 (ACD Bio# 323100)
- RNAScope Hydrogen Peroxide
- Protease Plus
- 50x Wash Buffer
- RNAScope Multiplex FL v2 AMP 1,2,3
- HRP-C1, HRP-C2, HRP-C3, HRP blocker
- TSA Buffer
- DAPI
- Akoya Biosciences Opal fluorophores (Opal 520 #FP1487001KT, Opal 570 #FP1488001KT, Opal 690 #FP1497001KT)
- HybEZ Hybridization System
- ProLong Gold Antifade Reagent from Life Technologies (P36930)
- RNase away and 70% EtOH for cleaning surfaces during section preparation (everything you use should be cleaned. Be careful of RNA degradation prior to fixation w PFA
Troubleshooting
Section Preparation
Perfuse mouse with 1xPBS and harvest brain (for younger mice extract brain without perfusing). ~7ml/min perfusion rate with pump. 1xPBS volume = weight (g) (ex. For 20g mouse, use 20ml of 1xPBS)
Immediately place brain into OCT mold, cover with OCT, and freeze in a metal beaker filled with 2-methylbutane, surrounded by dry ice in methanol.
Keep brain at -80C overnight. Section on the cryostat at 16 μm thickness onto SuperFrost Plus slides. Keep slides at -80C overnight.
Sample Fixation
Immerse slides in 4% PFA in 1xPBS, incubate slides for 2 hours (at least 15 min) at RT.
Wash x2 in 1xPBS
Dehydrate the Tissue
Immerse slides in 50% EtOH in DDW for 5 min at RT
Immerse slides in 70% EtOH in DDW for 5 min at RT
Immerse slides in 100% EtOH in DDW for 5 min at RT
Immerse slides in 100% EtOH in DDW for 5 min at RT
If necessary, store the slides in 100% EtOH at –20°C for up to 1 WEEK, but not recommended
Let slides dry and draw a barrier around sections at RT ~1 min
Make sure the hydrophobic barrier remains intact so that the tissues do not dry at any time.
Turn on HybEZ oven at 40C with Humidity Control Tray inside (mode RNAScope) to prepare for hybridization step. Place wet paper inside the tray.
Tissue Pretreatment
Add drops of RNAScope Hydrogen Peroxide. Incubate for 10 min at RT
Wash in DDW for 2 min x2
Add drops of Protease Plus. Incubate for 10 min at RT (100–150 µL is 4–6 drops)
While incubating, warm up your probes (warmed for 10 min at 40C, RT for 10 min) and 50x wash buffer. Precipitation occurs during storage.
Wash in 1xPBS for 2 min x2
Hybridization
Prepare probe master mix of C1:C2:C3 of 50:1:1. For ~4 sections per slide, ~100µL of C1, 2µL C2, 2µL C3. Include positive/negative control if possible.
Place slides locked in the Slide Holder into the Humidity Control Tray.
Add appropriate amount of probe mix to entirely cover each slide. Close tray and insert into oven for 2 hours at 40C.
While incubating:
1) Set a 1 hour 30 min timer to bring out the AMP boxes 30 min before the probe step
is over, to bring the AMPs to RT.
2) Prepare fresh 1x wash buffer (490ml DI water and 10ml of 50x wash buffer)
Wash in 1x wash buffer for 2 min x2
If necessary, STOPPING POINT – slides can be stored in 5x SSC buffer overnight at RT. Wash with 1x Wash buffer for 2 min at RT before continuing with assay, but not recommended
Amplification
Place slides onto the slide holder.
Apply AMP1 and incubate in oven for 30 min at 40C
Wash in 1x wash buffer for 2 min x2
Apply AMP2 and incubate in oven for 30 min at 40C
Wash in 1x wash buffer for 2 min x2
Apply AMP3 and incubate in oven for 15 min at 40C
Wash in 1x wash buffer for 2 min x2
During incubation: prepare 3 Opal secondaries – 1:750 in TSA buffer for Opal 520, 570, 690 and keep in dark. Equilibrate HRP-C1,-C2,-C3, and blockers to RT 30 min prior.
Develop HRP Signal
Apply HRP-C1 and incubate in oven for 15 min at 40C
Wash in 1x wash buffer for 2 min x2
Apply diluted fluorophore for C1 and incubate in oven for 30 min at 40C
Wash in 1x wash buffer for 2 min x2
Apply block and incubate in oven for 15 min at 40C
Wash in 1x wash buffer for 2 min x2
Repeat Steps 30-35 for C2 and C3 if using more channels.
Counterstain and Mount Slides
Apply DAPI and incubate for 30s - 5 min at RT
Decant DAPI and immediately cover slides with Prolong Gold
Coverslip slides with a 1.5 thickness slide cover
Let slides dry overnight in a dark place at 4C. Scan when fully dry.