Dec 11, 2022
Version 1
Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) in Drosophila CNS V.1
- Mark Eddison1,
- Gudrun Ihrke2
- 1Project Technical Resources, Janelia Research Campus, Ashburn, VA, USA.;
- 2HHMI Janelia Research Campus
- Project Technical Resources (PTR)
Protocol Citation: Mark Eddison, Gudrun Ihrke 2022. Expansion-assisted iterative fluorescence in situ hybridization (EASI-FISH) in Drosophila CNS. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jmw7g2w/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: September 28, 2022
Last Modified: December 11, 2022
Protocol Integer ID: 70604
Keywords: Drosophila CNS, In situ hybridization, HCR, Expansion Microscopy, EASI-FISH, iterative fluorescence in situ hybridization, fish protocol for adult drosophila cn, assisted iterative fluorescence, expansion microscopy, drosophila cn, adult drosophila cn, antibody detection of fluorescent reporter, situ hybridization, multiplexed in situ hybridization, fluorescence, hybridization chain reaction, drosophila, microscopy, antibody detection, thick slices of the mouse brain, mouse brain, fluorescent reporter, fluorescent, fish protocol
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 the EASI-FISH protocol for adult Drosophila CNS, which includes antibody detection of fluorescent reporters. The protocol also works well for larval CNS and is expected to be applicable to other tissue types.
Guidelines
Reference:
Wang et al., EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell184 (2021) 6361-6377.e24.
https://pubmed.ncbi.nlm.nih.gov/34875226/
Materials
Solutions for Fly brain dissection
- 2%PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEElectron Microscopy SciencesCatalog #15710 in S2 medium.
- Schneider's Drosophila MediumThermo FisherCatalog #21720024
- PBT (0.5% Triton).
- 70% Ethanol (in nuclease-free water).
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
- Stock-X. 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.
Stock X:
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
NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9760G
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 Stock X (not including APS +TEMED + 4HT):
| A | B | |
| 4.04M Sodium Acrylate | 2275 ul | |
| 40% Acrylamide | 625 ul | |
| 2% N,N MethylBisacrylamide | 750 ul | |
| 5M NaCl | 4000 ul | |
| 10x PBS | 1000 ul | |
| Nuclease Free Water | 750 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 % |
| A | B | |
| 1 M Tris> 50 mM | 2.5 ml | |
| 10 % Triton> 0.5 % | 2.5 ml | |
| 5 M NaCl> 50 mM | 0.5 ml | |
| 0.5 M EDTA | 100 ul | |
| 10% SDS > 0.3% SDS | 1.5 ml | |
| Nuclease Free Water | 42.9 ml |
Note
If brains have TdTomato as a reporter, in the buffer solution increase the NaCl to 500mM and remove SDS to preserve endogenous fluorescence. Depending on expression level, Myr-GFP fluorescence tends to withstand ProK digestion, but we also detect it with a directly conjugated GFP antibody to ensure a good signal.
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 |
Reagents for JF-669 conjugation to unlabelled hairpins:
- Janelia Fluor® 669TocrisCatalog #6420 Store -20 °C .
- 1 nanomolar (nM) unlabelled amine-modified hairpins (~70mer, Molecular Instruments, Store -20 °C ).
- Acetonitrile anhydrous Thermo Fisher ScientificCatalog #042311-K7
- 0.1 Molarity (M) Sodium Bicarbonate pH 8-9.
- DMSO, AnhydrousThermo FisherCatalog #D12345
- QIAquick Nucleotide Removal Kit (250)QiagenCatalog #28306
- SCREW CAP MICROCENTRIFUGE TUBES SELF-STANDING AMBER (0.5 ml)USA ScientificCatalog #1405-9707
Equipment
Equipment
Savant™ SpeedVac™ SPD120 Vacuum Concentrator and Kits
NAME
Vacuum Concentrator
TYPE
Savant
BRAND
SPD120-230
SKU
LINK
Troubleshooting
Fly brain dissection
Dissect fly CNS in S2 medium.
(for more details see attached protocol)
Fix up to 20 brains or 10 CNS in 2 mL of 2% PFA/S2 medium for 00:55:00 in the dark on a nutator.
55m
Rinse sample 1 x 2 mL PBST (0.5% Triton).
Wash sample 4 x 00:15:00 1 mL PBST(0.5%Triton) on a nutator.
15m
Rinse sample 1 x 2 mL 70% EtOH.
Store brains in 2 mL 70% EtOH @ 4 °C for up to 6 months.
Day 1: Labelling RNA
Transfer brains to a 0.2ml PCR tube (2-4 brains per 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 brains 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 30 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 brains 2 x 2 min 150 µL PBT and 1 x 2 mins 150 µL PBS.
Wash brains 2 x 00:02:00 150 µL PBT and 1 x 00:02:00 150 µL PBS.
4m
Wash brains 2 x 00:02:00 150 µL PBT and 1 x 00:02:00 150 µL PBS.
4m
Thaw Stock-X and 4HT, Temed and APS. Vortex well and keep On ice .
Gently stick down brains in the center of the chamber, once stuck down, carefully add a drop of PBS to prevent dehydration.
Note
It is possible to mount 4-5 brains per chamber.
Mix together Stock-X and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex.
Note
Each chamber needs ~120 µL of Stock-X, 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 brain, 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
DAPI stain gels for 00:10:00 with 1 mL DAPI/PBS (500 ng/ml).
10m
Rinse with PBS.
Use a dissection scope with a UV bulb to neatly trim gel edges with a razor blade.
Thaw and mix hybridization (hyb) and probe wash buffer. Make sure hyb buffer is clear.
Incubate gel in 500 µL hyb buffer for 00:30:00 @ 37 °C .
30m
Dilute probes 1/100 (10 Nanomolar (nM) ), 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 gels 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 .
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
Stain sample with 500 µL of anti-GFP-488 Ab @ (1/500) in PBT (0.1%) containing 5 mg/mL Ultrapure BSA and incubate Overnight (or the weekend) @ 4 °C .
15m
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 00:15:00 with 1 mL PBS/DAPI (500 ng/ml ).
15m
Mount gels (sample up) on an 8mm poly-lysine coated coverslip superglued to a Z.1 light-sheet sample holder and image.
For gel removal from the holder, incubate gels in 750 µL of 10% Dextran Sulphate for 20 mins. Gels will shrink and fall off the coverslip.
For long-term storage, keep gels in 750 µL 10% Dextran Sulphate @ 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).
JF-669 conjugation to unlabelled hairpins
1h 45m
Note
Hairpins are amine modified, JF-669 has an NHS ester group.
Turn on speed vacuum (eg. Thermofisher, SPD120) and defrost dye at Room temperature for 00:30:00 .
30m
Resuspend 2 mg of JF-669, SE in 630 µL Acetonitrile, mix and vortex, and aliquot in 30 µL into labelled skirted 0.5ml screw-cap centrifuge tubes > each will contain 0.1 mg of dye.
Evaporate acetonitrile in speed vacuum (in organic solvent mode) for 00:45:00 . Can store @ -20 °C .
45m
In two 1.5ml Eppendorf tubes, evaporate 5 µL (500 picomolar (pM) /10 µg ) of unlabelled hairpins h1 and h2 using a speed vac, in aqueous mode, for 00:30:00 . If you have 10 µL (1 nanomolar (nM) ) use two tubes per hairpin. Check they have been fully evaporated.
30m
Add 3 µL of 0.1 Molarity (M) Sodium Bicarbonate pH 8-9 to each evaporated hairpin. Mix.
Add 2 µL of anhydrous DMSO to 0.1 mg of dye. Mix.
Add 2 µL dye mix (100 µg ) to each 3 µL of hairpin (10 µg ). Mix. It will change colour.
Leave hairpin-dye mixture to react Overnight at Room temperature in the dark.
The next morning, add 5 µL nuclease-free water to bring to 10 µL .
Remove excess dye with a QIAquick Nucleotide removal kit (add 100 µL PN1).
Elute dye-oligo conjugate in 50 µL nuclease-free water.
Check the hairpin concentration on a spectrophotometer (eg. a NanoDrop One) and dilute to 60 ng/μl .
Separately store hairpins h1-669 and h2-669 in 25 µL aliquot’s in a PCR tube@ -20 °C .
Test conjugation by HCR.