Mar 13, 2025

Public workspaceExpansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) for morphoFISH

 Forked from Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) in Drosophila CNS
DOI
dx.doi.org/10.17504/protocols.io.5jyl824n6l2w/v1
  • 1Scientific Operations, Janelia Research Campus;
  • 2Janelia Research Campus;
  • 3Project Technical Resources, Janelia Research Campus
Mark Eddison
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DOI: dx.doi.org/10.17504/protocols.io.5jyl824n6l2w/v1
Protocol Citation: Monique Copeland, Tiago A 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
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.
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Mark Eddison
Materials


Solutions for Day 1: Labelling RNA

  • ReagentMOPS (Fine White Crystals/Molecular Biology)Fisher ScientificCatalog #bp308100.. Store TemperatureRoom temperature .

  • Melphalan-X (Stock Concentration2 mg/mL ; working Concentration1 mg/mL ). Store Temperature-20 °C .

  • ReagentAcryloyl-X, SEThermo Fisher ScientificCatalog #A20770 , Stock Concentration10 mg/mL ; working Concentration0.1 mg/mL . Store Temperature-20 °C .
  • ReagentPress-to-Seal™ Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 0.5 mm deepThermo FisherCatalog #P24743

  • ReagentPoly-L-Lysine solution 10mlTEDPELLACatalog #18026 , Amount1.6 mL +Amount3.2 µL ReagentPHOTO-FLO 200 SOLUTIONElectron Microscopy SciencesCatalog #74257 . Store Temperature4 °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 Temperature-20 °C .
  • 10% ReagentAmmonium PersulfateSigmaCatalog #A3678 . Store Temperature-20 °C .
  • 10% ReagentNNN′N′-TetramethylethylenediamineSigma AldrichCatalog #T22500 . Store Temperature-20 °C .
  • 0.5% Reagent4-Hydroxy-TEMPOSigmaCatalog #176141 ). Store Temperature-20 °C .
  • ReagentProteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S , Concentration800 U/ml ). Store Temperature-20 °C .
  • Concentration50 millimolar (mM) Proteinase K Buffer: Store TemperatureRoom temperature .
  • Small paint brush.


Solutions for Day 3: Hybridization

  • Hybridization Buffer (Molecular Instruments, Store Temperature-20 °C ).
  • Probe Wash Buffer (Molecular Instruments, Store Temperature-20 °C ).
  • DNA oligo probes (Designed and made by Molecular Instruments, Store Temperature-20 °C ).
  • DAPI (Sigma D9534)/ReagentPBSFisher ScientificCatalog #BP24384 at Concentration500 ng/ml .


Solutions for Day 5: Hybridization Chain Reaction (HCR)

  • Amplification Buffer (Molecular Instruments, Store Temperature4 °C ).
  • Fluorescent Hairpins (448 or 546 from Molecular Instruments or 669 conjugated in lab, Store Temperature-20 °C ).
  • 5x SSCT (5x SSC, 0.1% Tween in Nuclease Free Water). Store TemperatureRoom temperature .
  • 0.5x SSCT (0.5x SSC, 0.1% Tween in Nuclease Free Water). Store TemperatureRoom temperature .
  • ReagentInvitrogen GFP Polyclonal Antibody Alexa Fluor™ 488Thermo Fisher ScientificCatalog #A-21311 .
  • ReagentUltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog # AM2616..


Solutions for Stripping Probes and Hairpins for Multiplexing


ReagentDNAse I, RNAse-freeQiagenCatalog #79254 .
Note
Do not use RDD buffer supplied.

DNAse1 buffer

AB
Tris-HCL pH 8.010 mM
MgCl22.5 mM
CaCl20.5 mM


Recipes and Reagents


200mM MOPS Buffer (10X Stock):

Amount1046.5 mg in Amount25 mL in NFW, pH to 7.7 with Concentration10 Normality (N) NaOH. Store Temperature-20 °C .


Melphalan stock (2.5mg/ml)

ReagentMelphalanCayman Chemical CompanyCatalog #16665
ReagentDMSO, AnhydrousThermo FisherCatalog #D12345

  • Dissolve Amount2.5 mg per ml in anhydrous DMSO (Invitrogen, D12345).
  • Dissolve, heat to Temperature37 °C and vortex vigorously and place on a shaker.
  • May take an hour to dissolve.
  • Aliquot in Amount800 µL batches.
  • Store in a desiccated environment at Temperature-20 °C .


Acryloyl-X (AcX) stock (10 mg/ml)

ReagentAcryloyl-X, SEThermo Fisher ScientificCatalog #A20770

  • Dissolve in anhydrous DMSO.
  • Aliquot in Amount200 µL batches.
  • Any extra aliquot in Amount5 µL batches (for extra AcX).
  • Store in a desiccated environment at Temperature-20 °C .
  • Don’t re-use AcX after thawing.


Melphalan-X (2mg/ml):

  • Combine an equal concentration of Acryloyl-X (Concentration10 mg/mL ) and Melphalan (Concentration2.5 mg/mL ) (1-part AcX to 4-parts Melphalan (ie. Amount200 µL : Amount800 µL ).
  • Incubate DurationOvernight at TemperatureRoom temperature with shaking.
  • Store in Amount50 µL aliquots in a desiccated environment at Temperature-20 °C .
  • Use at Concentration1 mg/mL by 1:1 dilution with Concentration20 millimolar (mM) MOPS.


Trex-1000:

Concentration4.04 Molarity (M) Sodium Acrylate* – made from Acrylic Acid as it is made at variable purity
Reagent40% Acrylamide SolutionBio-rad LaboratoriesCatalog #1610140
Reagent2% Bis SolutionBio-rad LaboratoriesCatalog #1610142
ReagentNuclease-free water AmbionCatalog #AM9932
10X PBS (ThermoFisher; AM9625)

4.04M Sodium Acrylate stock solution

  • In a fume hood, place Amount5.5 mL of acrylic acid into a 50mL tube. Place tube in a TemperatureRoom temperature water bath (e.g. a beaker).
  • Add Amount4.5 mL water.
  • Add Amount7.2 mL Concentration10 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 Concentration1 Molarity (M) NaOH (nominally Amount1 mL ) gradually until the pH is between 7.5 and 8 using a pH meter, at TemperatureRoom temperature . Do not use pH test strips.
  • Add water up to a final volume of Amount20 mL .
Note
Acrylic acid has a pKa of 4.76 -- at Ph7.75 this solution has about Concentration4 millimolar (mM) remaining buffering capacity.



For 9.4 mls Trex-1000 (not including APS +TEMED + 4HT):

AB
4.04M Sodium Acrylate 2500 ul
40% Acrylamide 3571 ul
2% N,N MethylBisacrylamide500 ul
10x PBS1000 ul
Nuclease Free Water1829 ul


10% APS:
AB
APS100 mg
H20900 ul

10% Temed:
AB
TEMED100 ul
H20900 ul

0.5% 4HT:
AB
4HT5 mg
H20995 ul

Note
Store APS, TEMED and 4HT in Amount150 µL aliquots in PCR tubes @ Temperature-20 °C .



50mM ProK/SDS Buffer (50ml)

ReagentEDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
Reagent10% SDS solutionThermo Fisher ScientificCatalog #15553027

AB
Tris-HCL- pH850 mM
EDTA1 mM
TritonX0.5 %
NaCl50 mM
SDS0.3 %






RNase-Free DNase1

  • Add Amount550 µL DNAase1 Buffer to DNase1 powder. Mix.
  • Aliquot Amount50 µL per PCR tube, store @ Temperature-20 °C .

DNase1 buffer (50ml)

AB
1 M Tris-HCL > 10 mM500 ul
1 M MgCl2 > 2.5 mM125 ul
1 M CaCl2 > 0.5 mM25 ul
Nuclease Free Water49.350 ml


HCR Wash Buffers (50 ml)

5x SSCT:

ReagentSSC, RNase-free, 20×AmbionCatalog #AM9763

AB
20x RNase free-SSC (ThermoFisher, AM 9763)12.5 ml
10% Tween500 ul
Nuclease Free Water 37 ml


0.5x SSCT:
AB
20x RNase free-SSC1.25 ml
10% Tween 500 ul
Nuclease Free Water48.25 ml
































Preparation of slices for EASI-FISH
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
Day 1: Labelling RNA
Transfer each ROI slice to a 0.2ml PCR tube

Rehydrate with 2 x 5 min wash in Amount150 µL PBT (0.1%).

Pipetting
Wash
Rehydrate with 2 x Duration00:05:00 wash in Amount150 µL PBT (0.1%) (1/2).
5m
Rehydrate with 2 x Duration00:05:00 wash in Amount150 µL PBT (0.1%) (2/2).
5m
Incubate slice 1 x Duration00:30:00 in Amount150 µL Concentration20 millimolar (mM) MOPS Buffer.

30m
Incubation
Pipetting
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.
Pipetting
Dilute Melphalan-X stock 1:1 with MOPS Buffer (to 1mg/ml).
Add 1/100 Ac-X (Concentration10 mg/mL ) to Melphalan-X working solution. Vortex, mix, and spin.

Centrifigation
Pipetting
Mix
Add Amount50 µL of Melphalan-X/AcX solution to each PCR tube and gently mix.

Pipetting
Mix
Incubate DurationOvernight @ Temperature37 °C .

2m
Incubation
Overnight
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 Amount1 µL poly-Lysine using a P20 pipette tip. Air dry and repeat.
Download Chambers.jpgChambers.jpg

Pipetting
Day 2: Gelation and Proteinase K digestion
Day 2: Gelation and Proteinase K digestion
Wash slice 2 x 2 min Amount150 µL PBT and 1 x 2 mins Amount150 µL PBS.

Pipetting
Wash
Wash slice 2 x Duration00:02:00 Amount150 µL PBT and 1 x Duration00:02:00 Amount150 µL PBS.
4m
Wash slice 2 x Duration00:02:00 Amount150 µL PBT and 1 x Duration00:02:00 Amount150 µL PBS.
4m
Thaw TREx-1000 and 4HT, Temed and APS. Vortex well and keep TemperatureOn ice .

Mix
Gently stick down the slice in the center of the chamber, once stuck down, carefully add a drop of PBS to prevent dehydration.




Pipetting
Mix together Trex-1000 and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex.
Note
Each chamber needs ~Amount120 µL of Trex-1000, make excess. (ie. for two chambers make Amount300 µL gel solution).


Pipetting
Mix
Remove PBS from chamber with pipette tip and carefully wick away remaining PBS with a tissue.
Pipette Amount40 µL of gel solution, on top of the slice, to each chamber. Incubate slide in the fridge ( Temperature4 °C ) for Duration00:10:00 .

10m
Incubation
Pipetting
Take off gel solution and repeat step 22.

Note
Place waste gel solution in an Eppendorf. Before discarding, polymerize the gel waste @ Temperature37 °C



Take off the gel solution and gasket surface adhesive. Add a final Amount40 µL of gel solution and gently place a cover slip over the chamber. Gently press to seal the coverslip and incubate @ Temperature4 °C for Duration00:10:00 .

Note
Adding Amount5 µL of gel solution to the center of the underside of the coverslip can help prevent air bubbles when sealing.

10m
Incubation
Pipetting
Polymerize the gel @ Temperature37 °C for Duration01:30:00 to Duration02:00:00 .

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 Amount2 mL Eppendorf.

Incubate each gel with Amount1 mL ProK Buffer and Amount10 µL (1/100) ProK Enzyme @ Temperature37 °C DurationOvernight .

2h
Incubation
Pipetting
Overnight
Day 3: Hybridization
Day 3: Hybridization

Wash gels 4 x 15 min with Amount1 mL PBS at TemperatureRoom temperature .

Wash
Wash gels 4 x Duration00:15:00 in Amount1 mL PBS at TemperatureRoom temperature (1/4).
15m
Wash gels 4 x Duration00:15:00 in Amount1 mL PBS at TemperatureRoom temperature (2/4).
15m
Wash gels 4 x Duration00:15:00 in Amount1 mL PBS at TemperatureRoom temperature (3/4).
15m
Wash gels 4 x Duration00:15:00 in Amount1 mL PBS at TemperatureRoom 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.
Mix
Incubate gel in Amount500 µL hyb buffer for Duration00:30:00 @ Temperature37 °C .

30m
Incubation
Pipetting
Dilute probes 1/100 (Concentration10 Mass Percent ), in Amount300 µL hyb buffer per gel. Vortex. Incubate @ Temperature37 °C .
Incubation
Pipetting
Mix
Incubate gels with probes overnight @ Temperature37 °C , no shaking necessary.

Incubation
Put probe wash buffer and PBS @ Temperature37 °C .

Day 4: Probe Washing
Day 4: Probe Washing
Wash 3 x 30 min Amount750 µL Probe Wash Buffer @ Temperature37 °C .

Wash
Wash 3 x Duration00:30:00 Amount750 µL Probe Wash Buffer @ Temperature37 °C (1/3).

30m
Wash 3 x Duration00:30:00 Amount750 µL Probe Wash Buffer @ Temperature37 °C (2/3).
30m
Wash 3 x Duration00:30:00 Amount750 µL Probe Wash Buffer @ Temperature37 °C (3/3).
30m
Wash 3 x 30 min Amount1 mL PBS @ Temperature37 °C .

Wash
Wash 3 x Duration00:30:00 Amount1 mL PBS @ Temperature37 °C (1/3).

30m
Wash 3 x Duration00:30:00 Amount1 mL PBS @ Temperature37 °C (2/3).
30m
Wash 3 x Duration00:30:00 Amount1 mL PBS @ Temperature37 °C (3/3).
30m
Wash 3 x 1hr Amount1 mL PBS @ Temperature37 °C .

Wash
Wash 3 x Duration01:00:00 Amount1 mL PBS @ Temperature37 °C (1/3).
1h
Wash 3 x Duration01:00:00 Amount1 mL PBS @ Temperature37 °C (2/3).
1h
Wash 3 x Duration01:00:00 Amount1 mL PBS @ Temperature37 °C (3/3).
1h
Keep gels in PBS at TemperatureRoom temperature DurationOvernight .

1h
Overnight
Day 5: Hybridization Chain Reaction (HCR)
Day 5: Hybridization Chain Reaction (HCR)
Incubate gel in Amount500 µL Amplification buffer for at least Duration00:30:00 @ TemperatureRoom temperature

30m
Incubation
Pipetting
Snap cool hairpins with PCR machine @ Temperature95 °C for Duration00:01:30 and cool @ TemperatureRoom temperature for Duration00:30:00 .


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
PCR
Mix hairpins h1 and h2 @ 1/100 in Amount300 µL Amp Buffer per gel. Vortex.

Pipetting
Mix
Incubate gel with hairpins for Duration03:00:00 @ TemperatureRoom temperature in the dark.

3h
Incubation
Wash gels 2 x 20 min in Amount750 µL 5X SSCT @ TemperatureRoom temperature .

Wash
Wash gels 2 x Duration00:20:00 in Amount750 µL 5X SSCT @ TemperatureRoom temperature (1/2).
20m
Wash gels 2 x Duration00:20:00 in Amount750 µL 5X SSCT @ TemperatureRoom temperature (2/2).
20m
Wash gels 2 x 40 min in Amount1 mL 0.5X SSCT @ TemperatureRoom temperature .

Wash
Wash gels 2 x Duration00:40:00 in Amount1 mL 0.5X SSCT @ TemperatureRoom temperature (1/2).
40m
Wash gels 2 x Duration00:40:00 in Amount1 mL 0.5X SSCT @ TemperatureRoom temperature (2/2).
40m
Day 6: Mount and Image
Day 6: Mount and Image

Wash 2 x 30 min with Amount1 mL PBS-Triton (0.1%).

Wash
Wash 2 x Duration00:30:00 with Amount1 mL PBS-Triton (0.1%) (1/2).
30m
Wash 2 x Duration00:30:00 with Amount1 mL PBS-Triton (0.1%) (2/2).
30m
Wash 2 x 30 min and 1 x 1hr with Amount1 mL PBS.

Wash
Wash 2 x Duration00:30:00 with Amount1 mL PBS (1/2).
30m
Wash 1 x Duration01:00:00 with Amount1 mL PBS (2/2).
1h

DAPI stain gels for Duration01:00:00 with Amount1 mL PBS/DAPI (Concentration500 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.
Imaging
For long-term storage, keep gels in Amount750 µL 5X SSC @ Temperature4 °C

Stripping Probes and Hairpins for Multiplexing
Stripping Probes and Hairpins for Multiplexing
2h 45m
2h 45m

Incubate gel for Duration00:30:00 in Amount1 mL of DNAse1 Buffer @ Temperature37 °C .

30m
Incubation
Pipetting
Add Amount450 µL of DNase Buffer to Amount50 µL DNase1. Mix.

Pipetting
Mix
Incubate gel in DNase1 for Duration02:00:00 @ Temperature37 °C .

2h
Incubation
Wash 4 x 15 min with Amount1 mL PBS.

Wash
Wash 4 x Duration00:15:00 with Amount1 mL PBS (1/4).
15m
Wash 4 x Duration00:15:00 with Amount1 mL PBS (2/4).
15m
Wash 4 x Duration00:15:00 with Amount1 mL PBS (3/4).
15m
Wash 4 x Duration00:15:00 with Amount1 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