Feb 27, 2025

Public workspaceRNASCOPE In Situ Hybridization

This protocol is a draft, published without a DOI.
  • 1Karolinska Institutet;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Marta Graziano
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Protocol Citation: Marta Graziano, Ioannis Mantas, Konstantinos Meletis 2025. RNASCOPE In Situ Hybridization. protocols.io https://protocols.io/view/rnascope-in-situ-hybridization-d4ve8w3e
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: February 27, 2025
Last Modified: February 27, 2025
Protocol Integer ID: 123526
Abstract
Protocol for RNAScope ISH
Materials
Material preparations:

A) Material preparation:
- Place wet tissue or humidifying paper in the oven tray.
- Warm up HybEZ oven (Advanced Cell Diagnostics) to 40°C.
- Pre-heat everything for at least 20 minutes.
- Place amplifiers 1-4 at room temperature (RT).
- Make sure the washing case is clean.
- Make sure to have containers for waste/solutions.

B) Probe preparation:
- Warm up the probes (and if C1-channel is not filled, probe diluent) in the oven at 40°C for 10 minutes, then cool down to RT.
- Mix 50:1:1 ratios of chosen probes (50 volumes of C1, 1 volume of C2, and 1 volume of C3) in a tube and mix by inverting a couple of times.
- The mixed target probes can be stored at 4°C for 6 months.

C) Wash buffer:
- Prepare 1L of 1X wash buffer (Ref 310091): heat up 20mL pure wash buffer in the oven at 40°C for 10 minutes.
- Transfer the pure wash buffer into the 1L glass bottle and slowly add 980mL distilled water to it.
- This wash buffer can be kept for 1-2 months at RT.

D) Pretreat solution:
- Dilute pretreat2 (from RNAscope Target Retrieval Reagents; Ref 322000) in distilled water 1:10 to make preferred total volume.
- Boil the solution until soft boiling (99-100°C) is maintained, let cool down until 70-80°C.
FISH protocol (RNAscope Fluorescent Multiplex Assay V2)
FISH protocol (RNAscope Fluorescent Multiplex Assay V2)
Take samples from -80°C freezer and air dry them at RT, make sure to protect them from dust, make also sure that the bench you are working is clean.
For perfused brains:
Wash the samples in 1X PBS for approximately 5 minutes while gently moving the slides up and down to get rid of the OCT.
Boil Pretreat 2.
Transfer samples into the 70-80°C pretreat solution, leave them for 5 minutes.
Immediately transfer slides into wash case filled with distilled water, wash slides by moving up and down for 3-5 times. Repeat with fresh distilled water.
Quickly wash slides in 100% ethanol by moving up and down 3-5 times.
Air dry slides, again protect them from dust.
Once dry, draw a hydrophobic barrier with an immerge pen, let the barrier dry for approximately 2 minutes.
Place slides on HybEZ slide rack, add ca 1 drop Protease3 (from RNAScope H2O2 and Protease Reagents kit; Ref 322381) to the section. Add enough to cover the sections, make sure they do not dry out whilst being in the oven, but also do not overuse it. Incubate for 30 minutes at 40°C.
For fresh frozen sections:
Fix the sections in 4% PFA in 1xPBS DEPC for 15 minutes at 4°C.
Dehydrate the slides in 70%, 80%, 95%, and 100% ethanol, 2 minutes per each concentration.
Draw hydrophobic barrier with heat stable hydrophobic pen and let the slides and the pen dry.
Incubate the sections in Protease4 (from RNAScope H2O2 and Protease Reagents kit; Ref 322381) for 30 minutes at RT.
COMMON:
After incubation time, transfer the samples into the washing case containing distilled water and wash the slides by moving up and down 3-5 times. Repeat with fresh distilled water.
Following the next washing steps, tap and/or flick to remove excess liquid from slides and place them in HybEZ slide rack.
Add approximately 1 drop of appropriate probe or probe-mix to the samples so that the sections are covered completely. Incubate in the oven for 2 hours at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add approx. 1 drop AMP-1FL to each sample, so that the section is covered. Place in oven and incubate for 30 minutes at 40°C.
Quickly remove excess liquid by decanting. Place slides in washing case containing 1X wash buffer. Wash slides in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add approx. 1 drop AMP-2FL to each sample, so that the section is covered. Place in oven and incubate for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place slides in washing case containing 1X wash buffer. Wash slides in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add approx. 1 drop AMP-3FL to each sample, so that the section is covered. Place in oven and incubate for 30 minutes at 40°C.
Take out TSA-fluorophores from fridge (stored at 4°C), choose which color you want the channel to be and dilute in preferred volume (depending on gene expression level, if low expression is expected, try 1:500 first). Keep the diluted TSA-fluorophores in the dark, wrapped in aluminum foil.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add HRP Channel 1 (HRP-C1) to samples. Just enough to cover the sections. Place samples in the oven for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer, two times at RT. First wash quick, by moving up and down a couple of times. Second wash, 5 minutes.
Add preferred TSA-fluorophore to samples. Enough to cover the sections. Incubate for 5 minutes in the oven at 40°C.
If it is known that the gene has very low expression levels, incubate with TSA-fluorophore for 10 minutes and add a third washing cycle after the HRP-blocker step. This goes for all channels.
Take out the samples, remove excess liquid by decanting. Add HRP-blocker to the samples, enough to cover the sections. Place in the oven for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
If only one channel is being used, you can continue to the step of adding DAPI to the samples.
Add HRP-C2 to samples. Just enough to cover the sections. Place samples in the oven for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer, two times at RT. First wash quick, by moving up and down a couple of times. Second wash, 5 minutes.
Add preferred TSA-fluorophore to samples. Enough to cover the sections. Incubate for 5 minutes in the oven at 40°C.
Take out the samples, remove excess liquid by decanting. Add HRP-blocker to the samples, enough to cover the sections. Place in the oven for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add HRP-C3 to samples. Just enough to cover the sections. Place samples in the oven for 15 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer, two times at RT. First wash quick, by moving up and down a couple of times. Second wash, 5 minutes.
Add preferred TSA-fluorophore to samples. Enough to cover the sections. Incubate for 5 minutes in the oven at 40°C.
Take out the samples, remove excess liquid by decanting. Add HRP-blocker to the samples, enough to cover the sections. Place in the oven for 20 minutes at 40°C.
Quickly remove excess liquid by decanting. Place samples in washing case containing 1X wash buffer. Wash samples in wash buffer for 2 minutes at RT by moving up and down a couple of times. Repeat with fresh wash buffer.
Add DAPI to the samples, just enough to cover the sections. Let it incubate in a dark spot for 1-2 minutes.
Remove DAPI from slides and immediately place 1-2 drops of mounting medium onto each section.
Place cover slip on tissue section. Avoid trapping air bubbles. Store the slide in the dark at 4°C overnight before starting the confocal imaging.