Aug 21, 2025

Multiplex Detection of Gene Expression in the Intact Drosophila Brain using EASI-FISH V.2

DOI
https://dx.doi.org/10.17504/protocols.io.5jyl8jmw7g2w/v2
Multiplex Detection of Gene Expression in the Intact Drosophila Brain using EASI-FISH
  • 1Project Technical Resources, Janelia Research Campus, Howard Hughes Medical Institute
  • Project Technical Resources (PTR)
Mark Eddison
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DOI: https://dx.doi.org/10.17504/protocols.io.5jyl8jmw7g2w/v2
External link: http://link to PTR webpage
Protocol Citation: Kari Close, Yisheng He, Gudrun Ihrke, Mark Eddison 2025. Multiplex Detection of Gene Expression in the Intact Drosophila Brain using EASI-FISH. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jmw7g2w/v2Version created by Mark Eddison
Manuscript citation:
Kari Close, Yisheng He, Jennifer Jeter, Gudrun Ihrke, and Mark Eddison (2025). Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization. J. Vis. Exp. 219, e67656, doi:10.3791/67656

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: April 11, 2025
Last Modified: August 21, 2025
Protocol  Integer ID: 126606
Keywords: gene expression, in situ hybridization, mRNA, transcript, hybridization chain reaction, EASI-FISH, expansion microscopy, Drosophila melanogaster, Neuroscience, expansion microscopy with multiplexed fish, multiplex detection of gene expression, assisted iterative fluorescence in situ hybridization, green fluorescent protein, intact drosophila brain, assisted iterative fluorescence, gene expression, gene expression in thick tissue sample, multiplex detection, expansion microscopy, multiplexed fish, fish expansion, situ hybridization, fish protocol suitable for high throughput experimentation, several gel
Abstract
Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) is a powerful technique that integrates expansion microscopy with multiplexed FISH to investigate gene expression in thick tissue samples. We describe an updated fly EASI-FISH protocol suitable for high throughput experimentation. Up to six brains or two CNS can be embedded in a single gel and several gels can be processed in parallel. We use a new gel recipe that improves gel robustness and permits at least 10 rounds of hybridization. Using the GAL4-UAS system for co-detection of green fluorescent protein (GFP), gene expression can be visualized in cell types of interest. Due to the high resolution and sensitivity of the method, single transcripts can be detected, and spot counting can be used to quantify expression levels.
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Image Attribution
Kari Close, Yisheng He, Jennifer Jeter, Gudrun Ihrke, and Mark Eddison (2025). Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization. J. Vis. Exp. 219, e67656, doi:10.3791/67656
Materials

Solutions for Fly brain dissection

  • 4%PARAFORMALDEHYDE 16% Aqueous SOL. EM GRADEElectron Microscopy SciencesCatalog #15710 in PBS

  • 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

AB
Tris-HCL pH 8.010 mM
MgCl22.5 mM
CaCl20.5 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 Mass Percent 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 Mass Percent 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 Mass Percent 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):

AB
4.04M Sodium Acrylate2500 ul
40% Acrylamide 3571 ul
2% N,N MethylBisacrylamide500 ul
10x PBS1000 ul
Nucelase Free Water183 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 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

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

AB
1 M Tris> 50 mM2.5 ml
10 % Triton> 0.5 %2.5 ml
5 M NaCl> 50 mM0.5 ml
0.5 M EDTA100 ul
10% SDS > 0.3% SDS1.5 ml
Nuclease Free Water42.9 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)

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:

SSC, 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



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 Mass Percent 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
https://www.thermofisher.com/order/catalog/product/SPD120-230
LINK































Fly brain dissection
15m
Dissect fly CNS in cold S2 medium. Use steps that reduce chance of contamination with RNases: wear gloves, use RNase free reagents when available, clean bench and dissecting forceps with RNase away prior to use.
Fix up to 20 brains or 10 CNS in 2 mL of 4 °C 4% PFA/PBS medium Overnight in the dark on a nutator @ 4 °C. Transfer brains into cold fixative after each dissection to prevent RNA degradation.
The next day rinse sample 1 x 2 mL 4 °C PBT (0.5% Triton).

Wash sample 4 x 00:15:00 2 mL 4 °C PBT (0.5%Triton) on a nutator.

15m
Rinse sample 1 x 2 mL 4 °C 70% EtOH.

Store brains in 2 mL 70% EtOH @ 4 °C for up to 6 months.

Day 1: Mounting brains and adding RNA and protein anchors
20m
Prepare gel chambers by wiping non-charged slide with RNase away, coat slide twice with poly-lysine allowing to air dry between coating, preferably allowed to dry overnight after second coating.
The next day adhere silicone gasket with up to 4 chambers to slide.
Transfer brains from 70% EtOH to a 0.2ml PCR tube (2-4 brains per tube).

Rehydrate with 2 x 5 min wash in 150 µL PBT (0.1% Triton).


Rehydrate with 2 x 00:05:00 wash in 150 µL PBS.

Add 40ul of PBS per chamber and then mount brains by gently sticking them down in a row in the center of the chamber using fine tipped forceps. Note- Mounting up to 6 brains or 2 CNS per chamber is possible.
Remove PBS from chamber and add 50 µL 20 millimolar (mM) MOPS Buffer, incubate 1 x 00:30:00

While brains are equilibrating in MOPS buffer, thaw Melphalan-X (MelphX) and Acryloyl-X (Ac-X) solutions.
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. Note- time the preparation such that the solution is ready at the end of the 30 minute MOPS incubation.
Remove MOPS buffer and add 50 µL of Melphalan-X/AcX solution to each chamber.
Cover the chamber by gently placing a 25mm x 25mm coverslip on top, do not use gasket adhesive to seal.
Place slide in humidified box protected from light and incubate Overnight @ Room temperature



5m
Day 2: Gelation and Proteinase K digestion
2h 8m
The next day carefully remove the coverslips from the chambers and remove Melphalan-X/AcX solution.
Wash mounted brains in chambers 2 x 00:02:00 100 µL PBT (0.1% Triton).
4m
Wash mounted brains in chambers 2 x 00:02:00 100 µL PBS.
4m
Thaw TREx-1000 and 4HT, Temed and APS. Vortex well and keep On ice .

Mix together TREx-1000 and 4HT, Temed and APS at a ratio of 94:2:2:2. Vortex and keep on ice for the duration of the incubations @ 4 °C
Note
Each chamber needs ~120 µL of gel solution, make excess. (ie. for two chambers make 300 µL ).


Remove PBS from chamber with pipette tip and carefully wick away remaining PBS with a lint free wipe.
Pipette 40 µL of gel solution, on top of the brains, to each chamber. Incubate slide in the fridge ( 4 °C ) for 00:10:00 .

10m
Take off gel solution and add 40 µL of gel solution, on top of the brains, to each chamber. Incubate slide in the fridge ( 4 °C ) for another 00:10:00 .

Note
Place waste gel solution in an Eppendorf. Before discarding, polymerize the gel waste @ 37 °C



10m
Take off the gel solution and peel the clear plastic protective film off the gasket's surface adhesive. Add a final 45 µL of gel solution and gently place a coverslip over the chamber, avoiding trapping any air bubbles. Gently press to seal the coverslip and incubate @ 4 °C for another 00:10:00 (for a total of 30 minutes equilibration @ 4 °C

Note
Adding 5 µL of gel solution to the center of the underside of the coverslip can help prevent air bubbles when sealing.

10m
Remove slides from fridge and place in incubator to polymerize the gel @ 37 °C for 01:30:00 .

1h 30m
Once gels have set, allow to cool 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 500 µL ProK Buffer and 5 µL (1:100) ProK Enzyme @ 37 °C Overnight .

Day 3: DNA digestion and probe hybridization
4h 30m
The next day wash gels 3 x 10 min with 1 mL PBS @ Room temperature . Note- use a fine flexible tip pipette to avoid damaging the gels when changing solutions.
Note
Omit DNAse treatment before the first round of hybridization if nuclear DAPI staining needs to be preserved (e.g., for segmentation). In this case, samples should be washed with PBS 4x 15 min before proceeding.



30m
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. Gently mix but do not vortex
Incubate gel in DNase1 for 02:00:00 @ 37 °C .
2h
Wash 4 x 15 min with 1 mL PBS. Note- gels can be stored in 5x SSC @ 4 °C before or after Digest/DNase washes for several weeks.
1h
Thaw hybridization (hyb) buffer.
Incubate gel in 500 µL hyb buffer for 00:30:00 @ 37 °C .

30m
Dilute probes 1:100 in 300 µL hyb buffer per gel. Vortex.
Incubate gels with hybridization buffer containing probes overnight @ 37 °C , no shaking necessary.

Place probe wash buffer and PBS @ 37 °C for washes next day.

Day 4: Probe Washing
6h
The next day wash 3 x 30 min 750 µL Probe Wash Buffer @ 37 °C .

1h 30m
Wash 3 x 30 min 1 mL PBS @ 37 °C .

1h 30m
Wash 3 x 1 hr 1 mL PBS @ 37 °C .

3h
Keep gels in PBS @ Room temperature Overnight or @ 4 °C over the weekend.
Day 5: Hybridization Chain Reaction (HCR)
5h 46m 30s
Incubate gels in 500 µL Amplification buffer for at least 00:30:00 @ Room temperature

30m
In a PCR machine, heat the fluorescent hairpins to 95 °C for 00:01:30 and snap cool to 25 °C for 00:30:00 . Spin briefly to collect hairpins.

31m 30s
For each probe dilute matching hairpins h1 and h2 at 1:50 in 300 µL Amplification Buffer per gel. Vortex.

Incubate gel with hairpins for 03:00:00 @ Room temperature protected from light.

3h
Wash gels 2 x 15 min in 750 µL 5x SSCT @ Room temperature protected from light.

30m
Wash gels 2 x 30 min in 1 mL 0.5x SSCT @ Room temperature protected from light.

1h
If brains express nls-GFP to mark/and or segment cells of interest, stain sample with 500 µL of directly labeled anti-GFP-488 Ab (1:500) in PBT (0.1% Triton) containing 5 mg/mL Ultrapure BSA and incubate Overnight (or over the weekend) @ 4 °C .

15m
If brains do not require antibody staining, they can be kept in 1 mL PBS overnight or over the weekend @ 4 °C . Alternatively, they can be DAPI stained with 1 mL PBS/DAPI 5 µg/µL @ 4 °C overnight or over the weekend.
Day 6: Mount and Image
2h 30m
Prepare light sheet holder or glass bottom dish by coating with poly-lysine 2x allowing to dry @ 37 °C between coats.
If antibody stained wash 1 x 30 min with 1 mL PBT (0.1% Triton).
If no antibody stain skip to mounting steps.
30m
If antibody stained wash 3 x 30 min with 1 mL PBS.
1h 30m
If antibody stained, DAPI stain gel for at least 00:30:00 with 1 mL PBS/DAPI (5 µg/µL ).
30m
To mount gels remove most of PBS from tube and allow gel to slide out and place them gently onto a coverslip and carefully dry the edges with a lint free wipe.
Ensure the gel is oriented such that the tissue will be closest to the objective and using a paintbrush gently slide the gel onto the poly-lysine surface using one movement.
Return gel holder to 2ml tube and ensure the sample is submerged in PBS or alternatively bathe the glass bottom dish in PBS to prevent desiccation.
Gels will be expanded 2 x in PBS. For 3 x expansion wash gels in 0.05x SSC instead of PBS.

For gel removal gently slide paintbrush between surface of gel and gel holder or glass bottom dish, gels will easily dislodge. Transfer to 2ml tube for storage.

For long-term storage, keep gels in 1 mL 5x SSC @ 4 °C

For Cell Segmentation and Spot Quantification
1h
Omit DNAse treatment before the first round of hybridization. After step 32, samples should be washed with PBS 4 x 10 min with 1 mL PBS before proceeding to hybridization with a 28S ribosomal RNA probe (1 micromolar (µM) ) directly conjugated to a fluorophore (e.g., CF633) diluted 1:50.

After probe washing (Steps 43-45) stain with 1 mL PBS/DAPI (500 ng/ml ) for 01:00:00
1h
After imaging, cellular masks are created using segmentation software, utilizing the information from both the cytoplasmic stain (ribosomal RNA probe) and the nuclear DAPI stain to identify and outline individual cells. Fluorescent spots reporting single transcripts can be counted within these masks.
Note
We are currently developing a segmentation and spot counting protocol using CellposeSAM and fishspot, respectively; see additional information in the "References" section. Upon completion, this protocol will be made available at 10.17504/protocols.io.6qpvrwenzlmk/v1.

Stripping Probes and Hairpins for Multiplexing
4h 30m

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. Gently mix but do not vortex.

Incubate gel in DNase1 for 02:00:00 @ 37 °C .

2h
Wash 4 x 15 min with 1 mL PBS.

1h
Hybridize with next round of probes (Day 3, step 38).
CF633 Succinimidyl Ester conjugation to unlabeled hairpins
1h 45m

Note
647 dyes quickly photobleach in the gel. For a far red alternative, conjugate unlabelled hairpins to CF633 SE or Janelia Fluor 669 SE. Hairpins are amine modified, CF633/JF669 SE has an NHS ester group for conjugation.

Turn on speed vacuum (eg. Thermofisher, SPD120) and defrost dye @ Room temperature for 00:30:00 .

30m

Resuspend 1 micromolar (µM) of CF633, SE in 330 µL Acetonitrile, mix and vortex, and aliquot 30 µL into labelled skirted 0.5ml screw-cap centrifuge tubes. Each will contain ~ 0.08 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 unlabeled 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 molar Sodium Bicarbonate pH 8-9 to each evaporated hairpin. Mix.

Add 2 µL of anhydrous DMSO to 0.08 mg of dye. Mix well.

Add 2 µL of dye mix to each 3 µL of hairpin. Mix well.

Leave hairpin-dye mixture to react Overnight @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 75 µL nuclease-free water.

Check the hairpin concentration on a spectrophotometer (eg. a NanoDrop One) and dilute to 60 ng/μl .

Separately store hairpins h1-633 and h2-633 in 25 µL aliquot’s in a PCR tube @ -20 °C .

Test conjugation by HCR.
Protocol references
Kari Close, Yisheng He, Jennifer Jeter, Gudrun Ihrke, and Mark Eddison (2025). Multiplex Detection of Gene Expression in the Intact Drosophila Brain Using Expansion-Assisted Iterative Fluorescence In Situ Hybridization. J. Vis. Exp. 219, e67656, doi: 10.3791/67656

https://www.janelia.org/support-team/project-technical-resources/fly-easi-fish (The information on this page includes a video of the sample handling from step 7 to 60 in the protocol described here, as published in J. Vis. Exp. 219, e67656, doi: 10.3791/67656.)

Yuhan Wang, Mark Eddison, Greg Fleishman, Martin Weigert, Shengjin Xu, Tim Wang,Konrad Rokicki, Cristian Goina, Fredrick E. Henry, Andrew L. Lemire, Uwe Schmidt, Hui Yang, Karel Svoboda, Eugene W. Myers, Stephan Saalfeld, Wyatt Korff, Scott M. Sternson, and Paul W. Tillberg (2021). EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184:6361, doi: 10.1016/j.cell.2021.11.024

Claire Managan and Gudrun Ihrke (2025). EASI-FISH Spot Counting in Drosophila Brain Using RS-FISH. dx.doi.org/10.17504/protocols.io.ewov1opz7lr2/v1

Marius Pachitariu, Michael Rariden, Carsen Stringer (2025). Cellpose-SAM: superhuman generalization for cellular segmentation. bioRxiv preprint https://www.biorxiv.org/content/10.1101/2025.04.28.651001v1

Greg Fleishman (2023). Fishspot https://github.com/GFleishman/fishspot/blob/main/fishspot/detect.py

Sanna Koskela, Claire Managan, Carsen Springer, Mark Eddison, and Gudrun Ihrke. Quantification of gene expression in Drosophila brain by EASI-FISH spot counting. https://dx.doi.org/10.17504/protocols.io.6qpvrwenzlmk/v1 in progress
Acknowledgements
All work was performed at the Janelia Research Campus and supported by the Howard Hughes Medical Institute through funds to the MultiFISH team project and Project Technical Resources.