Aug 29, 2024
RNAScope in situ hybridization (ISH) multiplex fluorescent protocol
- Roberta Marongiu1,2,
- Santiago Unda1,2
- 1Department of Neurological Surgery, Weill Cornell Medical College, New York, NY 10065;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Roberta Marongiu, Santiago Unda 2024. RNAScope in situ hybridization (ISH) multiplex fluorescent protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb67o1lpk/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: January 26, 2022
Last Modified: September 24, 2024
Protocol Integer ID: 57433
Keywords: ASAPCRN, rnascope in situ hybridization, using rnascope kit, rnascope kit from acdbio company, rnascope, multiplex fluorescent protocol instruction, situ hybridization, fluorescent, acdbio company
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
Guidelines
ISH is used to detect RNA. Thus, it is very important that all the reagents, equipment, and bench space used are very clean and uncontaminated.
Materials
Reagents:
- RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850 - contains a pretreatment kit with RNAscope protease III RNAscope Protease IIIACDCatalog #322337 and RNAscope protease IV ; a detection kit (AMP1, AMP2, AMP3, HPR-C1, HPR-C2, HPR-C3, HPR blockers + DAPI solution) and wash buffer reagents (50X wash buffer)
- RNAscope Target Retrieval (x10)ACDCatalog #322001
- Prolong Gold Antifade MountantThermo Fisher ScientificCatalog #P36930
- RNAscope probes kit (probes + RNAscope probe diluent)
- Distilled water
Equipment:
- cover glass 24x50mm
- tubes (various sizes)
- paper towel - absorbent paper
- foil
- hydrophobic pen, ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
- slice rack
- slice holder
- water bath
- incubator
- oven (see below)
Equipment
HybEZ II Hybridization System (110 or 220V)
NAME
Oven
TYPE
ACDBio
BRAND
321711
SKU
- slice tray (see below)
Equipment
HybEZ Humidity Control Tray
NAME
ACDBio
BRAND
310012
SKU
Solution
20x SSC buffer stock
- Dissolve 175.3 g of NaCl and 88.2 g of sodium Citrate in 800 ml of ddH2O
- Adjust pH to 7.0 with a few drops of 1M HCl (~ 20 drops)
- Adjust volume to 1L with additional ddH2O (pH becomes ~7.06)
- Sterilize by autoclaving
-5x SSC dilution can be stored for up to 2 months at RT
320293_RNAscope Multiplex_UM_04102019.pdf 320535_TechNote_Fixed frozenFluorescent_2019.pdf Protocol materials
RNAscope Protease IIIACDCatalog #322337
Prolong Gold Antifade MountantThermo Fisher ScientificCatalog #P36930
RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850
RNAscope Target Retrieval (x10)ACDCatalog #322001
ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000
Troubleshooting
Before start
For Fixed Frozen Brain samples
The challenge with thicker sections (30 to 40 microns) is tissue attachment. Hence, there are additional fixation and baking steps with respect to the standard protocol.
- Cut 30 μm (or 40 μm) sections.
- Collect sections into 12-24 well plates in anti-freeze solution
- Store sections at -20oC for no more than 3-4 weeks; at 4°C for no more than 2 days.
For Fixed Frozen Nodose Ganglion samples
- Cut 14 to 20 μm sections in cryostat.
- Collect sections into SuperFrost Plus Gold slides, avoid more than 3 sections per slide, and OCT overlapping with tissue from a previous section.
- Let sections dry at room temperature for 1 hour.
- Store sections at -80oC for no more than 4-5 weeks.
Part 1 : Sample preparation for frozen Nodose ganglia samples
Select tissue – ~ 3 sections/sample.
Dry tissue at Room temperature for 01:00:00 to 02:00:00 .
Cover the slide loosely with foil to avoid contaminations.
3h
Wash in PBS 1X to remove OCT for 2X 00:15:00 .
15m
Part 1 : Sample preparation for frozen brain samples
Select tissue – ~ 3 sections/sample.
Wash in PBS 1X to remove agar or OCT for 2X 00:15:00 .
Wash free-floating sections into net-wells and black tray.
15m
Mount tissue on superfrost charged gold slides.
Dry at Room temperature for 01:00:00 - cover the slides loosely with foil to avoid contaminations.
1h
Rinse in ddH2O by moving slide rack up and down (3-5 times) for 3x 00:00:15 .
15s
Part 1 : Sample preparation
Dry at Room temperature for 01:00:00 - cover the slides loosely with foil to avoid contaminations.
1h
During this incubation, PRE-HEAT HYBRIDIZATION OVEN and SLIDE TRAY at 60 °C .
Bake at 60 °C for 01:00:00 .
1h
During this incubation, prepare 4% PFA and keep in the fridge 4 °C .
Post-fix in 4% PFA at 4 °C for 01:00:00 (in the fridge).
1h
During this time prepare the following buffers:
Make 1X target retrieval buffer RNAscope Target Retrieval (x10)ACDCatalog #322001 .
(Warm stock solution 10X in water bath at 40°C for ~10 min before diluting / 1 mL 10X stock solution + 9 mL ddH20 )
Make 1X wash buffer ( provided in RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850 ).
- Warm 50X stock solution in water bath at 40°C for 10-20 min before diluting
- 1mL 50X stock solution + 49 mL ddH20. MIX WELL!!!
- 3L of 1X wash buffer -> 60 mL 50x stock solution (1 bottle) + 2.94 L ddH20
- Store 1X wash buffer at RT for up to 1 month.
Prepare bottles with EtOH gradient solutions: 50%, 70%, and 100%.
Part 2: RNAscope pre-treatments
2h 8m 45s
*NOTE – After each step (most important after washes)- Remove excess liquid from the slides before adding new reagents by tapping or gently flicking the slide on absorbent paper.
If you have a hydrophobic barrier, you can also use a Kimwipe to absorb the left-over liquid. Tilting the slide slightly and allowing solutions to pool in one corner on the hydrophobic barrier to then dab with a Kimwipe works well.
Dehydration steps using EtOH gradient: 50%, 70%, 100%, new 100% - at Room temperature , 5 min/each.
TURN ON STEAMER and SETUP at 100 °C . Use thermometer to check temperature.
Dry at 60 °C for 00:15:00 .
15m
Add RNAscope hydrogen peroxide - incubate slides at Room temperature for 00:10:00 .
10m
If active bubbling happens, please extend the Room temperature incubation to 00:20:00 .
20m
15. Rinse in ddH2O by moving slide rack up and down (3-5 times) for 2 x 00:00:15 .
15s
Dry at 60 °C for 00:15:00 .
15m
PLACE JARS WITH ddH2O AND TARGET RETRIEVAL (TR) SOLUTION INTO STEAMER 00:15:00 before is needed. Before starting the target retrieval step make sure that the TR buffer is not actively bubbling.
15m
TR step in the steamer -> place slides in ddH2O jar for 15 sec then move them into TR solution jar for 00:10:00 100 °C .
10m
Rinse in ddH2O 00:00:15 by moving slide rack up and down (3-5 times).
15s
Dip in 100% EtOH at Room temperature for 00:03:00 .
3m
Dry 60 °C for 00:10:00 .
10m
Draw hydrophobic barrier around the tissue:
Using the hydrophobic pen, ImmEdge hydrophobic barrier pap penVector LaboratoriesCatalog #H-4000 , make a barrier as close to tissue as possible (without touching it) to reduce the amount of solution needed to cover the surface area.
While waiting for pen to dry, PRE-HEAT OVEN and SLIDE TRAY AT 40 °C .
Add (4-6) drops of protease III (provided in RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850 RNAscope Protease IIIACDCatalog #322337 ) , digest in humidity chamber at 40 °C for 00:30:00 .
30m
During protease incubation:
Equilibrate amplification reagents (provided in RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850 ) :
Remove AMP1, AMP2, AMP3, HPR-C1, HPR-C2, HPR-C3, HPR blockers from the fridge and equilibrate at RT.
Prepare the probes (provided in RNAscope Fluorescent Multiplex Reagent KitACDCatalog #320850 )
Warm probes and probe stock solution in water bath at 40 °C for 00:10:00
cool at Room temperature
Briefly spin probe stock solutions
Mix probes into a new tube - Probe ratios = C2:C3:C4:C1 = 1:1:1:50.
Example: 4μL C3 + 4μL C2 + 192 μL C1
If not using a C1 probe, use RNAScope probe diluent in its place
Invert tube several times to mix before use.
Store mixed probes at 2 °C -8 °C for up to 6 months.
10m
Rinse in ddH2O by moving slide rack up and down (3-5 times) for 2 x 00:00:15 .
15s
After this step, proceed IMMEDIATELLY to the RNAscope assay.
Part 3: RNAscope assay
3h 42m
PROBE HYBRIDIZATION
Add (4-6) drops of probe mixture to appropriate slide(s), add positive probe to positive control slide, add negative probe to negative control slide. Incubate at 40 °C for 02:00:00 .
2h
During this time prepare 5x SSC buffer:
1.25mL 20x SSC + 3.75mL ddH20
5x SSC buffer can be stored for up to 2 months at RT
Rinse with 1X wash buffer for 2x 00:02:00 .
2m
Cover tissue with 5x SSC, incubate at Room temperature Overnight in covered humidified chamber.
2m
DAY 2
TURN ON HYBRIDIZATION OVEN and setup it at 40 °C .
Remove 5x SSC solution.
Rinse with 1X wash buffer atRoom temperature for 00:02:00 .
2m
Add drops of Amp #1 at 40 °C for 00:30:00 (in humidified chamber into hybridization oven).
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of Amp #2 at 40 °C for 300:30:00 .
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of Amp #3 at 40 °C for 00:15:00 .
15m
During this time, dilute Opal fluorophores (range 1:750-1:3K; usually dil. 1:1500) in TSA buffer:
Determine volume needed (150-220 ul/slide).
Example: 0.5μL fluorophore in 750μL TSA buffer from kit
Use tinfoil to cover tubes with the solutions as fluorophores are light sensitive.
NOTE: if Opal 690 is assigned to C1 or C2, its concentration may need to be increased.
Rinse with 1X wash buffer atRoom temperature for 2x 00:02:00 .
2m
Add drops of HRP-C1 at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of diluted Opal selected for C1 at 40 °C for 00:30:00 .
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-blocker at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-C2 at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of diluted Opal selected for C2 at 40 °C for 00:30:00 .
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-blocker at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-C3 at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of diluted Opal selected for C3 at 40 °C for 00:30:00 .
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-blocker at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-C4 at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of diluted Opal selected for C4 at at 40 °C for 00:30:00 .
30m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
Add drops of HRP-blocker at 40 °C for 00:15:00 .
15m
Rinse with 1X wash buffer at Room temperature for 2x 00:02:00 .
2m
DAPI solution from RNAscope kit at Room temperature for 00:00:30 .
30s
Air dry slides briefly, covered (slides are light sensitive).
Coverslip with ProLong Gold Antifade Mountant (from Thermo Fisher) or other comparable fluorescent mountant.
Immediately store slides in a light-proof box at 4 °C Dry 30 min to O/N.
Do not leave out at RT.
To prolong the signal, clear nail polish may be used to seal the coverslip to the slide.
Image slides after 8 hours or within 2 weeks. Signal deteriorates fast.