Mar 13, 2025
Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) for morphoFISH
- Monique Copeland1,
- Tiago Ferreira2,
- Mark Eddison3
- 1Scientific Operations, Janelia Research Campus;
- 2Janelia Research Campus;
- 3Project Technical Resources, Janelia Research Campus
Protocol Citation: Monique Copeland, Tiago Ferreira, Mark Eddison 2025. Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) for morphoFISH. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl824n6l2w/v1
Manuscript citation:
Discovery of neuronal cell types by pairing whole cell reconstructions with RNA expression profiles
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: October 09, 2024
Last Modified: March 13, 2025
Protocol Integer ID: 109476
Keywords: In situ hybridization, HCR, Expansion Microscopy, EASI-FISH, MorphoFISH, Mice, iterative fluorescence in situ hybridization, expansion microscopy, thick slices of the mouse brain, assisted iterative fluorescence, multiplexed in situ hybridization, situ hybridization, mouse slices with gfp, mouse brain, cleared mouse slice, labeled neuron, microscopy, hybridization chain reaction, modified gel recipe, gel recipe
Funders Acknowledgements:
HHMI
Grant ID: N/A
Abstract
A straightforward, robust, and reliable protocol (EASI-FISH) that utilizes expansion microscopy and the hybridization chain reaction for multiplexed in situ hybridization in thick slices of the mouse brain has recently been described (Wang et al., 2021). Below details a modified version of this protocol for cleared mouse slices with GFP-labeled neurons fixed under morphoFISH prefusion (Ferreira et al., 2024). This uses a modified gel recipe (TREx-1000) for greater gel stability. We also include an updated design for the Z1 sample holder.
Image Attribution
Mark Eddison
Materials
Solutions for Day 1: Labelling RNA
- MOPS (Fine White Crystals/Molecular Biology)Fisher ScientificCatalog #bp308100.. Store Room temperature .
- Melphalan-X (Stock 2 mg/mL ; working 1 mg/mL ). Store -20 °C .
- Acryloyl-X, SEThermo Fisher ScientificCatalog #A20770 , Stock 10 mg/mL ; working 0.1 mg/mL . Store -20 °C .
- Press-to-Seal™ Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 0.5 mm deepThermo FisherCatalog #P24743
- Poly-L-Lysine solution 10mlTEDPELLACatalog #18026 , 1.6 mL +3.2 µL PHOTO-FLO 200 SOLUTIONElectron Microscopy SciencesCatalog #74257 . Store 4 °C .
- PBS-Triton (0.1%)
- 0.2 ml PCR tubes (USA Scientific; 1402-4700).
- RNase Away (Thermo Scientific; 7003).
Solutions for Day 2: Gelation and Proteinase K digestion
- Trex-1000. Store -20 °C .
- 10% Ammonium PersulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A3678 . Store -20 °C .
- 10% NNN′N′-TetramethylethylenediamineMerck MilliporeSigma (Sigma-Aldrich)Catalog #T22500 . Store -20 °C .
- 0.5% 4-Hydroxy-TEMPOMerck MilliporeSigma (Sigma-Aldrich)Catalog #176141 ). Store -20 °C .
- Proteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S , 800 U/ml ). Store -20 °C .
- 50 millimolar (mM) Proteinase K Buffer: Store Room temperature .
- Small paint brush.
Solutions for Day 3: Hybridization
- Hybridization Buffer (Molecular Instruments, Store -20 °C ).
- Probe Wash Buffer (Molecular Instruments, Store -20 °C ).
- DNA oligo probes (Designed and made by Molecular Instruments, Store -20 °C ).
- DAPI (Sigma D9534)/PBSFisher ScientificCatalog #BP24384 at 500 ng/ml .
Solutions for Day 5: Hybridization Chain Reaction (HCR)
- Amplification Buffer (Molecular Instruments, Store 4 °C ).
- Fluorescent Hairpins (448 or 546 from Molecular Instruments or 669 conjugated in lab, Store -20 °C ).
- 5x SSCT (5x SSC, 0.1% Tween in Nuclease Free Water). Store Room temperature .
- 0.5x SSCT (0.5x SSC, 0.1% Tween in Nuclease Free Water). Store Room temperature .
- Invitrogen GFP Polyclonal Antibody Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-21311 .
- UltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616..
Solutions for Stripping Probes and Hairpins for Multiplexing
DNAse I, RNAse-freeQiagenCatalog #79254 .
Note
Do not use RDD buffer supplied.
DNAse1 buffer
| A | B | |
| Tris-HCL pH 8.0 | 10 mM | |
| MgCl2 | 2.5 mM | |
| CaCl2 | 0.5 mM |
Recipes and Reagents
200mM MOPS Buffer (10X Stock):
1046.5 mg in 25 mL in NFW, pH to 7.7 with 10 Normality (N) NaOH. Store -20 °C .
Melphalan stock (2.5mg/ml)
MelphalanCayman Chemical CompanyCatalog #16665
DMSO, AnhydrousThermo FisherCatalog #D12345
- Dissolve 2.5 mg per ml in anhydrous DMSO (Invitrogen, D12345).
- Dissolve, heat to 37 °C and vortex vigorously and place on a shaker.
- May take an hour to dissolve.
- Aliquot in 800 µL batches.
- Store in a desiccated environment at -20 °C .
Acryloyl-X (AcX) stock (10 mg/ml)
Acryloyl-X, SEThermo Fisher ScientificCatalog #A20770
- Dissolve in anhydrous DMSO.
- Aliquot in 200 µL batches.
- Any extra aliquot in 5 µL batches (for extra AcX).
- Store in a desiccated environment at -20 °C .
- Don’t re-use AcX after thawing.
Melphalan-X (2mg/ml):
- Combine an equal concentration of Acryloyl-X (10 mg/mL ) and Melphalan (2.5 mg/mL ) (1-part AcX to 4-parts Melphalan (ie. 200 µL : 800 µL ).
- Incubate Overnight at Room temperature with shaking.
- Store in 50 µL aliquots in a desiccated environment at -20 °C .
- Use at 1 mg/mL by 1:1 dilution with 20 millimolar (mM) MOPS.
Trex-1000:
4.04 Molarity (M) Sodium Acrylate* – made from Acrylic Acid as it is made at variable purity
40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140
2% Bis SolutionBio-Rad LaboratoriesCatalog #1610142
Nuclease-free water AmbionCatalog #AM9932
10X PBS (ThermoFisher; AM9625)
4.04M Sodium Acrylate stock solution
- In a fume hood, place 5.5 mL of acrylic acid into a 50mL tube. Place tube in a Room temperature water bath (e.g. a beaker).
- Add 4.5 mL water.
- Add 7.2 mL 10 Molarity (M) NaOH gradually to prevent excessive heating and evaporation/boiling.
- Remove tube from hood (at this point most of the acrylic acid has been converted to non-volatile sodium acrylate).
- Add 1 Molarity (M) NaOH (nominally 1 mL ) gradually until the pH is between 7.5 and 8 using a pH meter, at Room temperature . Do not use pH test strips.
- Add water up to a final volume of 20 mL .
Note
Acrylic acid has a pKa of 4.76 -- at 7.75 this solution has about 4 millimolar (mM) remaining buffering capacity.
For 9.4 mls Trex-1000 (not including APS +TEMED + 4HT):
| A | B | |
| 4.04M Sodium Acrylate | 2500 ul | |
| 40% Acrylamide | 3571 ul | |
| 2% N,N MethylBisacrylamide | 500 ul | |
| 10x PBS | 1000 ul | |
| Nuclease Free Water | 1829 ul |
10% APS:
| A | B | |
| APS | 100 mg | |
| H20 | 900 ul |
10% Temed:
| A | B | |
| TEMED | 100 ul | |
| H20 | 900 ul |
0.5% 4HT:
| A | B | |
| 4HT | 5 mg | |
| H20 | 995 ul |
Note
Store APS, TEMED and 4HT in 150 µL aliquots in PCR tubes @ -20 °C .
50mM ProK/SDS Buffer (50ml)
EDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
10% SDS solutionThermo Fisher ScientificCatalog #15553027
| A | B | |
| Tris-HCL- pH8 | 50 mM | |
| EDTA | 1 mM | |
| TritonX | 0.5 % | |
| NaCl | 50 mM | |
| SDS | 0.3 % |
RNase-Free DNase1
- Add 550 µL DNAase1 Buffer to DNase1 powder. Mix.
- Aliquot 50 µL per PCR tube, store @ -20 °C .
DNase1 buffer (50ml)
| A | B | |
| 1 M Tris-HCL > 10 mM | 500 ul | |
| 1 M MgCl2 > 2.5 mM | 125 ul | |
| 1 M CaCl2 > 0.5 mM | 25 ul | |
| Nuclease Free Water | 49.350 ml |
HCR Wash Buffers (50 ml)
5x SSCT:
SSC, RNase-free, 20×AmbionCatalog #AM9763
| A | B | |
| 20x RNase free-SSC (ThermoFisher, AM 9763) | 12.5 ml | |
| 10% Tween | 500 ul | |
| Nuclease Free Water | 37 ml |
0.5x SSCT:
| A | B | |
| 20x RNase free-SSC | 1.25 ml | |
| 10% Tween | 500 ul | |
| Nuclease Free Water | 48.25 ml |
Troubleshooting
Preparation of slices for EASI-FISH
To visualize the GFP neurons, rehydrate the slice in PBS for 10 mins.
Under a dissection scope and using a sharp knife, cut ROI with GFP cells from brain slice. The area should be about 2mm x 4mm and have a nick in the right hand corner to keep track of orientation. Take a photo of before and after to keep track of slice and cells.
Store ROI slice in 70% EtOH until ready to use
Day 1: Labelling RNA
Transfer each ROI slice to a 0.2ml PCR tube
Rehydrate with 2 x 5 min wash in 150 µL PBT (0.1%).
Rehydrate with 2 x 00:05:00 wash in 150 µL PBT (0.1%) (1/2).
5m
Rehydrate with 2 x 00:05:00 wash in 150 µL PBT (0.1%) (2/2).
5m
Incubate slice 1 x 00:30:00 in 150 µL 20 millimolar (mM) MOPS Buffer.
30m
Thaw Melphalan-X (MelphX) and Acryloyl-X (Ac-X) solutions.
Using a P20 pipette, take off as much MOPS buffer from the brains as possible.
Dilute Melphalan-X stock 1:1 with MOPS Buffer (to 1mg/ml).
Add 1/100 Ac-X (10 mg/mL ) to Melphalan-X working solution. Vortex, mix, and spin.
Add 50 µL of Melphalan-X/AcX solution to each PCR tube and gently mix.
Incubate Overnight @ 37 °C .
2m
Prepare gel chambers for gelation the next day. Wipe a non-charged slide with RNase away. Adhere a gasket (4-6 wells max) and coat the glass surface of the chamber with 1 µL poly-Lysine using a P20 pipette tip. Air dry and repeat.
Chambers.jpg Day 2: Gelation and Proteinase K digestion
Wash slice 2 x 2 min 150 µL PBT and 1 x 2 mins 150 µL PBS.
Wash slice 2 x 00:02:00 150 µL PBT and 1 x 00:02:00 150 µL PBS.
4m
Wash slice 2 x 00:02:00 150 µL PBT and 1 x 00:02:00 150 µL PBS.
4m
Thaw TREx-1000 and 4HT, Temed and APS. Vortex well and keep On ice .
Gently stick down the slice in the center of the chamber, once stuck down, carefully add a drop of PBS to prevent dehydration.
Mix together Trex-1000 and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex.
Note
Each chamber needs ~120 µL of Trex-1000, make excess. (ie. for two chambers make 300 µL gel solution).
Remove PBS from chamber with pipette tip and carefully wick away remaining PBS with a tissue.
Pipette 40 µL of gel solution, on top of the slice, to each chamber. Incubate slide in the fridge ( 4 °C ) for 00:10:00 .
10m
Take off gel solution and repeat step 22.
Note
Place waste gel solution in an Eppendorf. Before discarding, polymerize the gel waste @ 37 °C
Take off the gel solution and gasket surface adhesive. Add a final 40 µL of gel solution and gently place a cover slip over the chamber. Gently press to seal the coverslip and incubate @ 4 °C for 00:10:00 .
Note
Adding 5 µL of gel solution to the center of the underside of the coverslip can help prevent air bubbles when sealing.
10m
Polymerize the gel @ 37 °C for 01:30:00 to 02:00:00 .
3h 30m
Cool gels on the bench for a few minutes.
Take off the chamber lid and gasket with a razor blade. Trim the gels into a rectangle and nick the top right-hand corner to track the orientation of the sample.
Take off the gel from the slide with a paintbrush that has been wetted with a small amount of ProK Buffer and transfer it to a 2 mL Eppendorf.
Incubate each gel with 1 mL ProK Buffer and 10 µL (1/100) ProK Enzyme @ 37 °C Overnight .
2h
Day 3: Hybridization
Wash gels 4 x 15 min with 1 mL PBS at Room temperature .
Wash gels 4 x 00:15:00 in 1 mL PBS at Room temperature (1/4).
15m
Wash gels 4 x 00:15:00 in 1 mL PBS at Room temperature (2/4).
15m
Wash gels 4 x 00:15:00 in 1 mL PBS at Room temperature (3/4).
15m
Wash gels 4 x 00:15:00 in 1 mL PBS at Room temperature (4/4).
15m
DNAse treat sample before hybridization (see Step 48) for Cyto-DAPI staining/segmentation
Thaw and mix hybridization (hyb) and probe wash buffer.
Incubate gel in 500 µL hyb buffer for 00:30:00 @ 37 °C .
30m
Dilute probes 1/100 (10 Mass Percent ), in 300 µL hyb buffer per gel. Vortex. Incubate @ 37 °C .
Incubate gels with probes overnight @ 37 °C , no shaking necessary.
Put probe wash buffer and PBS @ 37 °C .
Day 4: Probe Washing
Wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C .
Wash 3 x 00:30:00 750 µL Probe Wash Buffer @ 37 °C (1/3).
30m
Wash 3 x 00:30:00 750 µL Probe Wash Buffer @ 37 °C (2/3).
30m
Wash 3 x 00:30:00 750 µL Probe Wash Buffer @ 37 °C (3/3).
30m
Wash 3 x 30 min 1 mL PBS @ 37 °C .
Wash 3 x 00:30:00 1 mL PBS @ 37 °C (1/3).
30m
Wash 3 x 00:30:00 1 mL PBS @ 37 °C (2/3).
30m
Wash 3 x 00:30:00 1 mL PBS @ 37 °C (3/3).
30m
Wash 3 x 1hr 1 mL PBS @ 37 °C .
Wash 3 x 01:00:00 1 mL PBS @ 37 °C (1/3).
1h
Wash 3 x 01:00:00 1 mL PBS @ 37 °C (2/3).
1h
Wash 3 x 01:00:00 1 mL PBS @ 37 °C (3/3).
1h
Keep gels in PBS at Room temperature Overnight .
1h
Day 5: Hybridization Chain Reaction (HCR)
Incubate gel in 500 µL Amplification buffer for at least 00:30:00 @ Room temperature
30m
Snap cool hairpins with PCR machine @ 95 °C for 00:01:30 and cool @ Room temperature for 00:30:00 .
Note
Choice of far-red dyes: We found that JF669 tends to bleach faster in morphoFISH tissue. For this reason, we prefer to use AF647-conjugated hairpins.
31m 30s
Mix hairpins h1 and h2 @ 1/100 in 300 µL Amp Buffer per gel. Vortex.
Incubate gel with hairpins for 03:00:00 @ Room temperature in the dark.
3h
Wash gels 2 x 20 min in 750 µL 5X SSCT @ Room temperature .
Wash gels 2 x 00:20:00 in 750 µL 5X SSCT @ Room temperature (1/2).
20m
Wash gels 2 x 00:20:00 in 750 µL 5X SSCT @ Room temperature (2/2).
20m
Wash gels 2 x 40 min in 1 mL 0.5X SSCT @ Room temperature .
Wash gels 2 x 00:40:00 in 1 mL 0.5X SSCT @ Room temperature (1/2).
40m
Wash gels 2 x 00:40:00 in 1 mL 0.5X SSCT @ Room temperature (2/2).
40m
Day 6: Mount and Image
Wash 2 x 30 min with 1 mL PBS-Triton (0.1%).
Wash 2 x 00:30:00 with 1 mL PBS-Triton (0.1%) (1/2).
30m
Wash 2 x 00:30:00 with 1 mL PBS-Triton (0.1%) (2/2).
30m
Wash 2 x 30 min and 1 x 1hr with 1 mL PBS.
Wash 2 x 00:30:00 with 1 mL PBS (1/2).
30m
Wash 1 x 01:00:00 with 1 mL PBS (2/2).
1h
DAPI stain gels for 01:00:00 with 1 mL PBS/DAPI (500 ng/ml ).
1h
Mount gels (sample up) on an 8mm poly-lysine coated coverslip superglued to a Z.1 light-sheet sample holder and image.
For long-term storage, keep gels in 750 µL 5X SSC @ 4 °C
Stripping Probes and Hairpins for Multiplexing
2h 45m
Incubate gel for 00:30:00 in 1 mL of DNAse1 Buffer @ 37 °C .
30m
Add 450 µL of DNase Buffer to 50 µL DNase1. Mix.
Incubate gel in DNase1 for 02:00:00 @ 37 °C .
2h
Wash 4 x 15 min with 1 mL PBS.
Wash 4 x 00:15:00 with 1 mL PBS (1/4).
15m
Wash 4 x 00:15:00 with 1 mL PBS (2/4).
15m
Wash 4 x 00:15:00 with 1 mL PBS (3/4).
15m
Wash 4 x 00:15:00 with 1 mL PBS (4/4).
15m
Hybridize with next round of probes (Day 3, step 34).
Protocol references
Wang et al. (2021) EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184 6361-6377.e24, https://pubmed.ncbi.nlm.nih.gov/34875226
Hugo GJ et al. (2022) Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx). eLife 11:e73775, https://doi.org/10.7554/eLife.73775
Ferreira AT et al. (2024) Discovery of neuronal cell types by pairing whole cell reconstructions with RNA expression profiles. bioRxiv 2024.12.30.630829, https://doi.org/10.1101/2024.12.30.630829