Jan 09, 2026
Version 5
WHOLISTIC ExM: Whole-Body Expansion Microscopy with Immunofluorescence and Histological Stains V.5
- Virginie M S Ruetten1,
- Amy Hu2,
- Mark Eddison2,
- Kari Close2,
- Yisheng He2,
- Misha B. Ahrens2,
- Paul Tillberg2
- 1Janelia Research Campus, HHMI - Gatsby Computational Neuroscience Unit;
- 2HHMI/Janelia Research Campus
Protocol Citation: Virginie M S Ruetten, Amy Hu, Mark Eddison, Kari Close, Yisheng He, Misha B. Ahrens, Paul Tillberg 2026. WHOLISTIC ExM: Whole-Body Expansion Microscopy with Immunofluorescence and Histological Stains. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9wxjvzp/v5Version created by Virginie M S Ruetten
Manuscript citation:
bioarxiv preprint
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 26, 2025
Last Modified: January 09, 2026
Protocol Integer ID: 228281
Keywords: Expansion Microscopy, Zebrafish, immunohistochemistry, clearing, WHOLISTIC, body expansion microscopy with immunofluorescence, body expansion microscopy, internal anatomy of larval zebrafish, resolution atlas of the larval zebrafish brain, using expansion microscopy, expansion microscopy method, quantitative ultrastructural analysis in whole vertebrate embryo, larval zebrafish brain, nanoscale resolution imaging of the whole mouse embryo, expansion microscopy, zebrafish brain atlas, subcellular resolution, wide cellular resolution imaging, whole vertebrate embryo, larval zebrafish, microscopy method, fish for thick tissue define, whole body imaging, microscopy, advanced whole body expansion, wide cellular resolution imaging of cre transgenic, anatomical data with enhanced processing speed, zebrafish line, double transgenic zebrafish animal, zebrafish, zebrafish lines for functional circuit, anatomical data, type information at subcellular resolution, whole mouse embryo, interpretation of whole body imaging, nano
Abstract
The interpretation of Whole Body Imaging (WBI) data necessitates comprehensive anatomical knowledge to accurately determine cell-type identity; however, resources pertaining to the internal anatomy of larval zebrafish are limited. To mitigate this gap, we established an advanced Whole Body Expansion-Microscopy (WB-ExM) protocol, facilitating the acquisition of molecular and cell-type information at subcellular resolution throughout the entire organism. While high-quality resources are available for the larval zebrafish brain1,2,3 there remains a deficiency in materials concerning its visceral anatomy. In order to supplement existing histological, X-ray, and Expansion Microscopy methods aimed at mapping body-wide anatomy4,5,6,7 and to develop a technique that yields high-fidelity molecular and anatomical data with enhanced processing speeds and compatibility with older samples, we devised an enzyme-free, rapid, and robust whole-body expansion microscopy method8,9, effective on larvae up to at least 14 days post-fertilization. This method involves the use of high-temperature (100°C) chemical hydrolysis to uniformly soften tissues, embedding within a medium-density gel with reduced protein-gel anchoring, and repeated embedding post-digestion with moderate ~1.5x expansion factors in each cycle, culminating in a robust and uniform expansion even of challenging structures such as cartilage embedded in soft tissue. This protocol results in excellent optical clearing, retains high levels of antibody signals.
A demo dataset can be viewed here. Dorsal view of a double transgenic zebrafish animal labeling the ventricular and vascular systems (Tg(foxj1a:eGFP) x Tg(flk1:dsRed-CAAX)), stained against eGFP (magenta) and dsRed (green) (10 days post-fertilization, expanded ~2×).
1. Kunst, M. et al. A Cellular-Resolution Atlas of the Larval Zebrafish Brain. Neuron 103, 21-38.e5 (2019).
2. Randlett, O. et al. Whole-brain activity mapping onto a zebrafish brain atlas. Nat Methods 12, 1039–1046 (2015).
3. Tabor, K. M. et al. Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping.
4. Copper, J. E. et al. Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 208, 38–46 (2018).
5. Ding, Y. et al. Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography. eLife 8, e44898 (2019).
6. Steib, E. et al. TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. Cell Reports Methods 2, 100311 (2022).
7. Sim, J. et al. Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy. Preprint at https://doi.org/10.1101/2021.05.18.443629 (2021).
8. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy.
9. Wang, Y. et al. EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184, 6361-6377.e24 (2021).
Image Attribution
Virginia M. S. Ruetten
Protocol materials
Corning® 25x25 mm Square #2 Cover GlassCorningCatalog #2855-25
CS-8R Coverslips, 0.15 mm (0.006 in), 8 mm diameter, pkg of 100Multi Channel Systems MCS GmbHCatalog #640701
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeThermo Fisher ScientificCatalog #AM9625
Pierce™ 16% Formaldehyde (w/v), Methanol-freeThermo ScientificCatalog #28906
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Normal Goat SerumJackson ImmunoResearch Laboratories, Inc.Catalog #005-000-121
PBS, pH 7.4Thermo FisherCatalog #10010001
Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-5ML
Sodium AzideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-100G
Agarose, low gelling temperatureMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9414-100G
Fisherbrand™ Superfrost™ Disposable Microscope SlidesFisher ScientificCatalog #Catalog No.12-550-123
Press-to-Seal™ Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 0.5 mm deepThermo Fisher ScientificCatalog #P24743
Poly-L-lysine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #Poly-L-lysine hydrobromide
Photo Flo 200 SolutionElectron Microscopy SciencesCatalog #74257
Alexa Fluor™ 488 NHS Ester (Succinimidyl Ester)Thermo Fisher ScientificCatalog #A20000
ATTO 647N maleimideAAT BioquestCatalog #2857
DMSO, AnhydrousThermo FisherCatalog #D12345
anti RFP antibodySynaptic SystemsCatalog #409 006
Anti-Rabbit-IgG - Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #40839-1ML-F
Donkey anti-Chicken IgY (H L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor™ 568Thermo Fisher ScientificCatalog #A78950
GFP Polyclonal AntibodyInvitrogen - Thermo FisherCatalog #A-11122
Acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid, succinimidyl ester)Thermo Fisher ScientificCatalog #A20770
Hydrogen peroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H1009-500ML
NaCl (5 M), RNase-freeThermo Fisher ScientificCatalog #AM9760G
UltraPure™ SDS Solution, 10%Thermo Fisher ScientificCatalog #15553027
Acrylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230-5G
Sodium Hydroxide Solution (10N/Certified)Fisher ScientificCatalog #SS255-1
N,N,N′,N′-Tetramethylethylenediamine (TEMED)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7024-25ML
2% bis-acrylamide solutionBio-Rad LaboratoriesCatalog #1610142
Ammonium persulfate (APS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3678-100G
40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140
Acrylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230-500G
4-Hydroxy-TEMPO (4HT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #4-Hydroxy-TEMPO
Before start
Make sure you have all the reagents at hand.
Reagents
4% PFA
Cannot be prepared in advance.
Take a new ampule of 16% PFA (10ml). Aliquot it into 1 ml aliquots. Store at -80ºC. On the day take one aliquot out and thaw.
To make a total volume of 4 ml of 4% PFA: mix 1ml of 16% PFA, 0.4 ml of 10x PBS and 2.6 ml of distilled H20. Use within the next two days and store in a 4ºC fridge.
Note
PFA deteriorates even if stored at -80ºC. Avoid reusing aliquoted samples.
When PFA is stored in sealed ampules, it is protected from atmospheric oxygen and moisture. This isolation prevents oxidative degradation
Reagents
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeThermo Fisher ScientificCatalog #AM9625
Pierce™ 16% Formaldehyde (w/v), Methanol-freeThermo ScientificCatalog #28906
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Blocking Buffer
Cannot be prepared in advance.
5% goat serum, 0.5% Triton-100 in 1x PBS, 0.1% Na Azide.
Keep in fridge at 4ºC
4 °C
To make 7.5 ml of Blocking Buffer
- 0.375 ml of goat serum
- 0.75 ml of PBS-T-5-NaAz-1 [PBS + Triton (5%) + NaAzide (1%)]
- 6.375 ml of 1x PBS
Note
Notes on Goat Serum:
This comes in a sealed vial. Do not open the seal but use a syringe to extract medium. The medium is nutrient rich and so easily can become contaminated. Do not use for more than 3 months. Reconstitute with distilled water.
Reagents
Normal Goat SerumJackson ImmunoResearch Laboratories, Inc.Catalog #005-000-121
PBS, pH 7.4Thermo FisherCatalog #10010001
Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-5ML
Sodium AzideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2002-100G
Antibodies
Primary antibodies (polyclonal)
rabbit anti-eGFP
chicken anti-RFP
anti-mRuby
Secondary antibodies
goat anti-rabbit Atto647N
donkey anti-chicken Alexa568
GFP Polyclonal AntibodyInvitrogen - Thermo FisherCatalog #A-11122
anti RFP antibodySynaptic SystemsCatalog #409 006
Anti-Rabbit-IgG - Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #40839-1ML-F
Donkey anti-Chicken IgY (H L) Highly Cross Adsorbed Secondary Antibody, Alexa Fluor™ 568Thermo Fisher ScientificCatalog #A78950
PBST-0.5
Can be prepared in advance and stored at room temperature.
1x PBS with 0.5% or 0.1% Triton
PBST-0.5 and PBST-0.1 respectively
Triton dissolves terribly. It hardens upon making contact with water and takes time to dissolve fully. Prepare a 10% stock solution in 1x PBS and use that for subsequent rounds.
Keep at RT.
Room temperature
Reagents
PBS, pH 7.4Thermo FisherCatalog #10010001
Triton™ X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100-5ML
Bleaching Solution
Cannot be prepared in advance.
Combine 3% H2O2, 60mM KOH and 100mM (or more) NaN3 in PBS.
- Prepare 300mM NaN3: add 195mg of NaN3 to 10ml of PBS.
To make 5 ml of bleaching solution:
- 0.3 ml of 50% H2O2 solution
- 0.1 ml of 1M KOH solution (takes one hour with little bubbling) [tested]
- fill the rest of 100mM NaN3 (i.e.: 4.25ml, 85mM NaN3)
Note
Many biological tissues contain catalase, which rapidly breaks down hydrogen peroxide into water and oxygen. This leads to a large number of bubbles, which can rupture the tissue. The tissue should first be passivated. NaN3 is a catalase inhibitor. Pre-incubate fish with NaN3 to block catalase. Then add bleach solution containing NaN3. The dissociation of H2O2 occurs faster at high pH, thus higher KOH should've used. The concentration of NaN3 is in excess to saturate the reaction - can be lowered.
Hydrogen peroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H1009-500ML
1% low-melting temperature agarose
Can be prepared in advance.
Dissolve 1 g of low-melting point agarose in 100 ml of 1x PBS.
Add 1 g of power to 100 ml 1x PBS at RT. Stir with magnetic stirrer.
Bring to a boil using a microwave. Stir with magnetic stirrer until full dissolved.
Repeat boil and stirring until solution is clear.
Reagents
Agarose, low gelling temperatureMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9414-100G
PBS - Phosphate-Buffered Saline (10X) pH 7.4, RNase-freeThermo Fisher ScientificCatalog #AM9625
Acryloyl-X Solution (AcX)
Cannot be prepared in advance.
Stock concentration: 10 mg/ml
Dissolve to 10 mg/ml in anhydrous DMSO. Aliquot in 20 µl batches.
Store in a desiccated environment at -20ºC. Don’t re-use AcX after thawing.
Working solution will be: 20 µg/ml, dilution 1:500 in 1x PBS
Note
Using anhydrous DMSO is important because DMSO is remarkably hygroscopic, readily absorbing water vapor from ambient air. DMSO-water mixtures have different solvation properties than pure DMSO, which can cause precipitation of hydrophobic compounds. The presence of water also enables hydrolysis of susceptible functional groups, degrading stock solutions over time.
Reagents
Acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid, succinimidyl ester)Thermo Fisher ScientificCatalog #A20770
DMSO, AnhydrousThermo FisherCatalog #D12345
Na Acrylate Solution
Fill a 500 ml beaker with ~300 ml of water for a water bath.
Place an open 50 ml Eppendorf tube into the bath and bring the setup to a fume hood.
The purpose of the water bath is to provide a highly conductive medium to cool the solution.
Add 9.0 ml of molecular grade water to the Eppendorf tube.
Add 11 ml of pure acrylic acid to the Eppendorf tube. (Note that this is a flammable and reactive compound).
Add 14.4 ml of 10 M NaOH to the Eppendorf tube. This should be done dropwise to prevent excessive heating and boiling.
Leave it to cool.
The solution should be clear. If a yellow precipitate is observed this means the chemicals need to be changed.
Calibrate a pH meter.
Remove the acrylate-filled tube from the fume hood.
By now, most of the acrylic acid will have been converted to non-volatile sodium acrylate.
Measure the pH of the solution.
Add NaOH to adjust the pH gradually to between 7.5-8 using a pH meter. Do NOT use pH test strips.
We recommend starting by adding 10 M NaOH solution, and when the pH gets close to the desired pH adjusting via the use of 1 M NaOH solution.
(As a general guidance: Add about 500 µl 10 M NaOH, 250 µl 10 M NaOH, 120 µL 10 M NaOH, then some 1 M NaOH)
Add water up to a final volume of 40 ml.
(Note: Acrylic acid has a pKa of 4.76 at pH 7.75 - this solution has about 4 mM remaining buffering capacity)
Acrylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230-5G
Sodium Hydroxide Solution (10N/Certified)Fisher ScientificCatalog #SS255-1
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Monomer and Gelation Solutions #1 (Medium Density Gel with high Bis)
| A | B | C | D | E | F | |
| name | units | stock conc. | final conc. | vol (ul) | vol (ml) | |
| Acrylamide | % | 40 | 10 | 250 | 2.5 | |
| Na Acrylate | M | 4 | 0.5 | 125 | 1.25 | |
| Bis | % | 1 | 0.1 | 100 | 1 | |
| 10x PBS | x | 10 | 1 | 100 | 1 | |
| Water | 365 | 3.65 |
Monomer Solution #1
Monomer Solution #1 can be stored safely at -20ºC for 1-3 months.
| A | B | C | D | E | |
| name (units) | units | stock conc. | final conc. | vol (ul) | |
| Monomer Solution #1 | 940 | ||||
| APS | % | 10 | 0.2 | 20 | |
| TEMED | % | 10 | 0.2 | 20 | |
| 4HT | % | 0.5 | 0.01 | 20 |
Gelation Solution #1
Reagents
40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140
2% bis-acrylamide solutionBio-Rad LaboratoriesCatalog #1610142
Acrylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230-500G
Sodium Hydroxide Solution (10N/Certified)Fisher ScientificCatalog #SS255-1
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Ammonium persulfate (APS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3678-100G
4-Hydroxy-TEMPO (4HT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #4-Hydroxy-TEMPO
N,N,N′,N′-Tetramethylethylenediamine (TEMED)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7024-25ML
Monomer and Gelation Solutions #2 (Medium Density Gel with low Bis)
| A | B | C | D | E | F | |
| to make 1ml | to make 10ml | |||||
| component | units | stock conc. | final conc. | vol (ul) | vol (ml) | |
| Acrylamide | % | 40 | 10 | 250 | 2.5 | |
| Na Acrylate | M | 4 | 0.5 | 125 | 1.25 | |
| Bis | % | 1 | 0.02 | 20 | 0.2 | |
| 10x PBS | x | 10 | 1 | 100 | 1 | |
| Water | 445 | 4.45 | ||||
Monomer Solution #2
Monomer Solution #2 can be stored safely at -20ºC for 1-3 months.
| A | B | C | D | E | |
| to make 1ml | |||||
| component | units | stock conc. | final conc. | vol (ul) | |
| Monomer Solution #2 | 940 | ||||
| APS | % | 10 | 0.2 | 20 | |
| TEMED | % | 10 | 0.2 | 20 | |
| 4HT | % | 0.5 | 0.01 | 20 | |
Gelation Solution #2
Reagents
40% Acrylamide SolutionBio-Rad LaboratoriesCatalog #1610140
2% bis-acrylamide solutionBio-Rad LaboratoriesCatalog #1610142
Acrylic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230-5G
Sodium Hydroxide Solution (10N/Certified)Fisher ScientificCatalog #SS255-1
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Ammonium persulfate (APS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #A3678-100G
4-Hydroxy-TEMPO (4HT)Merck MilliporeSigma (Sigma-Aldrich)Catalog #4-Hydroxy-TEMPO
N,N,N′,N′-Tetramethylethylenediamine (TEMED)Merck MilliporeSigma (Sigma-Aldrich)Catalog #T7024-25ML
Disruption Buffer
| A | B | C | D | E | F | |
| to make 1ml | to make 100ml | |||||
| component | units | stock conc. | final conc. | vol (ul) | vol | |
| SDS | % | 10 | 5 | 0.5 | 50 | |
| Tris pH 7.5 | mM | 1000 | 50 | 0.05 | 5 | |
| NaCl | M | 5 | 0.2 | 0.04 | 4 | |
| MilliQ water | 0.41 | 41 |
Disruption Buffer
Stock Disruption Buffer can be prepared in advanced and stored at RT for a few months.
NaCl (5 M), RNase-freeThermo Fisher ScientificCatalog #AM9760G
UltraPure™ SDS Solution, 10%Thermo Fisher ScientificCatalog #15553027
UltraPure™ DNase/RNase-Free Distilled WaterThermo Fisher ScientificCatalog #10977023
Total Protein Stains
Prepare stock: 10 mg/ml stocks in anhydrous DMSO.
Aliquot in 5-10ul.
Store in a desiccated environment at -20ºC.
Reagents
Alexa Fluor™ 488 NHS Ester (Succinimidyl Ester)Thermo Fisher ScientificCatalog #A20000
ATTO 647N maleimideAAT BioquestCatalog #2857
DMSO, AnhydrousThermo FisherCatalog #D12345
Gelation Chambers
Silicone gaskets (Invitrogen P24743)
Glass slides (SuperFrost 12550123)
Scotch tape
Poly-l-lysine
Shaker (nutator - 75 RPM)
Reagents
Press-to-Seal™ Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 0.5 mm deepThermo Fisher ScientificCatalog #P24743
Fisherbrand™ Superfrost™ Disposable Microscope SlidesFisher ScientificCatalog #Catalog No.12-550-123
Poly-L-lysine hydrobromideMerck MilliporeSigma (Sigma-Aldrich)Catalog #Poly-L-lysine hydrobromide
Photo Flo 200 SolutionElectron Microscopy SciencesCatalog #74257
General note for sample handling
Soak plastic transfer pipettes in PBST-0.5 before manipulating samples - otherwise, the fish will inevitably get stuck.
Never use forceps to handle or move the fish as this inevitably damages the sample.
Fixation and Permeabilization
10h
Euthanize samples with an overdose of MS-222 (a.k.a. tricaine) (200-300 mg/L).
Prepare fresh 4% PFA.
Place samples in 1 ml of 4% PFA.
Room temperature
Keep overnight (~9h) in 4% PFA at 4ºC on a shaker.
09:00:00
4 °C
Note
Fixation time can be adjusted by an hour or two as convenient. Over-fixation causes excessive cross-linking, which masks epitopes and makes tissue stiff. Under-fixation results in poorer protein retention.
9h
Rinse samples in 4 x 15 min 1 ml 1x PBS.
01:00:00
Room temperature
1h
Bleaching
1h
Prepare fresh Bleaching Solution
Combine 3% H2O2, 0.5% KOH (~90mM) and 50mM (or more) NaN3 in PBS.
Prepare 100mM NaN3:
Add 65mg of NaN3 to 10ml of PBS.
NaN3 is toxic so use a fume hood.
Lower concentrations of NaN3 would likely be acceptable too. We aimed at saturating the reaction.
To make 5 ml of Bleaching Solution:
- 0.3 ml of 50% H2O2 solution
- 0.45 ml of 1M KOH solution (0.3ml is still quite aggressive)
- fill the rest of 100mM NaN3 (i.e.: 4.25ml) to get to 85mM of 0.5% KOH (~90mM) and 50mM (or more) NaN3 in PBS.
Note
NB: Bleaching is not a required step, and users should decide whether to include it. If using Casper fish, for instance, bleaching will ensure the eyes are void of pigments, but have little other benefit. Bleaching comes with the risk of micro bubbles being retained in the tissue and so if it does needed we recommend avoiding it.
Hydrogen peroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H1009-500ML
Hydrogen peroxide solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #H1009-500ML
Preincubate the samples in 100mM NaN3 (~10mins).
Incubate the samples in 500 µL of Bleaching Solution.
Some bubbling will occur, but less is better. Leave the fish in the solution until eye pigments are visibly reduced (light brown), which should take approximately 30 min at RT. If pigments clear faster, the step can be stopped early. While we have not noticed any detrimental effects of 40 min incubation on antigenicity, shorter incubations are preferable as H₂O₂ can damage epitopes.
Room temperature
00:50:00
50m
Rinse the samples with 1x PBS (2x 5 min at RT).
Room temperature
00:10:00
10m
Immunofluorescence (Optional)
5d 17h 10m
Note
If immunohistochemistry is desired, we strongly recommend performing it prior to expansion. The protocol uses high-temperature hydrolytic disruption to homogenize the tissue, which denatures proteins and tends to destroy epitopes. Post-expansion immunohistochemistry will be supported in future versions of the protocol.
Prepare fresh Blocking Buffer.
Incubate the samples in Blocking Buffer (3 hr at RT) on a shaker.
Room temperature
03:00:00
3h
Incubate the samples in primary antibodies diluted 1:100 in Blocking Buffer in PCR tube (3 days at RT) on a shaker. Make sure Na Azide is included in the Blocking Buffer to prevent microbial growth.
72:00:00
Room temperature
Note
Can I place multiple samples in the same PCR tube?
Yes, multiple samples can be placed in the same PCR tube (~10). The level of antibody is high enough that it should not get significantly depleted.
What antibodies can I use?
As we do IHC pre-expansion and digestion, any antibody that works well in fixed whole-mount preparation should work in expansion.
3d
Wash sample in 1 ml Blocking Buffer (4 x 15 min at RT) on a shaker.
Room temperature
01:00:00
1h
Wash the samples in Blocking Buffer (3 x 2 hr or overnight at RT) on a shaker.
Room temperature
09:00:00
9h
Incubate the samples in secondary antibodies (1:100) in Blocking Buffer in PCR tube (2 days at RT) on a shaker.
48:00:00
Room temperature
2d
Wash the samples in Blocking Buffer (3x 2 hr, at RT).
Room temperature
04:00:00
4h
Wash the samples in 1x PBS (2x 5 min at RT) and transfer to fresh 1x PBS.
Room temperature
00:10:00
10m
Image one sample to confirm immunofluorescence staining was successful.
Permeabilization (if no IHC)
5h 10m
For specimens that have not been stained with antibodies, permeabilize in PBST-0.5 at 4ºC overnight (or for at least 5 hr).
05:00:00
4 °C
5h
Wash samples (2x 5 min at RT) in 1x PBS.
Room temperature
00:10:00
10m
Agarose Embedding
Heat agarose in microwave until it liquifies.
Cool agarose to 50ºC (by placing in 50ºC incubator).
Note
NB: Agarose embedding is not a mandatory step. Agarose embedding facilitates mounting the samples in a consistent orientation, which can greatly ease downstream data analysis and so is recommended, but not necessary.
Adhere silicone gasket (Invitrogen P24743 or Invitrogen P24740) on a glass slide (Superfrost Microscope Slides #12550123) to form a Mounting Chamber.
Mounting Chamber
Reagents
Press-to-Seal™ Silicone Isolator with Adhesive, eight wells, 9 mm diameter, 0.5 mm deepThermo Fisher ScientificCatalog #P24743
Fisherbrand™ Superfrost™ Disposable Microscope SlidesFisher ScientificCatalog #Catalog No.12-550-123
Place fish in Mounting Chamber, remove any access PBS, and cover with ~70-100 µl of 1% low-melting-point agarose. Orientate as desired (on side or dorsal side up) and leave to solidify.
Note
Ensure that the fish if fully covered in agarose (both above and below it). It can sometimes be easier to add an excess of agarose to make sure fish is fully immersed, and then remove the excess. The agarose should fill the chamber while not doming much.
If the sample isn't fully immersed, this can lead to the fish sticking to the slide.
Note
Use a sharp and clean utensil to nudge the fish into the desired orientation.
If more time is needed, the mounting chamber can be placed on a heating block to ensure that the agarose doesn't solidify pre-emptively.
Always begin by nudging the fish to the very bottom of the agarose drop to avoid the sample being held by surface tension to the top of the drop. Make sure that the fish is fully submerged in agarose.
With a razor blade, cut out a rectangle around the agarose-embedded sample and delicately lift the pad of agarose and transfer it to 12 well-plate with 1 ml of 1x PBS/well.
Cut as close to the fish as possible with some safety margin.
Note
To facilitate lifting of the agarose-embedded agarose, immersing the slide in 1x PBS can help. Ensuring that the sample is fully immersed in agarose also facilitates this step (see comments above).
Protein Anchoring
1h
Prepare Acryloyl-X Solution (stock solution 10 mg/ml, working solution: 20 µg/ml, dilution 1:500 in 1x PBS).
1 ml per sample is needed.
Do not prepare in advance.
Incubate each sample in 1 ml of Acryloyl-X Solution (1 hr at RT) shaking in 12-well plate.
Room temperature
01:00:00
1h
Preparation of Gelation Chamber #1
Prepare chambers for gelation, one for each sample.
Layer 11 pieces of Scotch tape together to create ~0.6 mm thick spacers.
Cut two strips of spacer material, about 2 cm long and 0.5 cm wide.
Stick two strips to a glass slide ~15 mm apart to form the side walls of the Gelation Chamber #1.
Gelation Chamber #1
Incubation with Gelation Solution 1
45m
Rinse samples with 1x PBS (3x 5 min) at RT.
00:15:00
Room temperature
15m
Thaw Monomer Solution #1, as well as 4HT, TEMED and APS. Vortex well and keep on ice.
Monomer Solution #1 and #2 can be stored safely at -20ºC for a few months.
Mix Monomer Solution #1 and 4HT, TEMED and APS at a ratio of 94:2:2:2 to produce Gelation Solution #1.
Vortex.
For reference: Each sample needs ~3 ml of Gelation Solution #1 (~1 ml per incubation round).
Incubate samples in Gelation Solution #1 on ice (3x 10 min at 4ºC) with 1 ml of Gelation Solution #1 on a shaker in a 12-well plate.
00:30:00
4 °C
30m
Gelation 1
2h
With spatula, transfer agarose block containing fish into the Gelation Chamber #1.
Gently place cover slip over the agarose block lying on the walls of Gelation Chamber #1 (made of scotch tape).
The cover slip should lay flat on the agarose block.
Gently press to seal.
Slowly pipette Gelation Solution #1 into Gelation Chamber #1 until full. Avoid any air bubbles.
Solution will hold by water tension.
One can use Gelation Solution #1 that was used during the previous incubation (no need to prepare fresh one).
Note
Avoiding bubbles is critical. If bubbles appear, the procedure should be repeated (cover slip removed and repositioned) until no bubbles are present. Bubbles contain oxygen, which inhibits polymerization by scavenging the free radicals that drive the chain reaction. Moreover, bubbles can become trapped within the gel and cause distortions and artifacts during imaging. Bubbles further than ~1mm away from the sample are acceptable though should still be minimized.
Once Gelation Chamber #1 is filled, place the chambers at 37ºC for 2 hr to induce polymerization.
Ensure incubator is humidified to prevent gel from drying out. This can be achieved by placing a beaker of water in the incubator.
37 °C
02:00:00
Note
Once polymerized, acrylamide forms non-toxic polyacrylamide, though gels may initially contain trace residual monomer so washing them thoroughly is recommended.
2h
Disruption
9h 5m
Turn on the 100ºC heat block (for later disruption step).
Prepare Disruption Buffer (5 ml/sample).
Stock Disruption Buffer can be prepared in advanced and stored at RT for a few months (e.g.: 50-100 ml stock). The solution should be clear. If precipitates form in the solution, this is a sign that it should have been re-made.
Place gels at RT, and allow them to cool on the bench for > 5 min.
Room temperature
00:05:00
5m
Take off coverslip lid with a razor blade.
Under a stereomicroscope, trim the gels into a rectangle with a ~1-3 mm border on either side of the sample.
Add a nick on the top right corner to be able to track orientation of the samples.
The gel should be about 9 mm x 4 mm.
Transfer each gel to a 2 ml Eppendorf tube and add 1.8 ml of Disruption Buffer and leave for 10 min to dilute any unpolymerized residues.
Remove the Disruption Buffer and fill with fresh Disruption Buffer.
Incubate each samples in Disruption Buffer (overnight at 100ºC).
Make sure that lid is closed tightly to avoid evaporation.
100 °C
09:00:00
After disruption, the gel should be about 12.5 mm x 6 mm.
9h
Chamber 2 preparation
Prepare Gelation Chamber #2, one for each sample.
Layer 20 pieces of Scotch tape together to create ~1.2 mm thick spacers.
Cut two strips of spacer material, about 2 cm long and 0.5 cm wide.
Stick two strips to a glass slide ~15 mm apart to form the side walls of the Gelation Chamber #2.
Incubation with Gelation Solution 2
1h 30m
Wash each samples in 1x PBS at RT (3x 20 min).
Room temperature
01:00:00
1h
Thaw Monomer Solution #2, as well as 4HT, TEMED and APS. Vortex well and keep on ice.
Monomer Solution #1 and #2 can be stored safely at -20ºC for a few months.
Mix Monomer Solution #2 and 4HT, TEMED and APS at a ratio of 94:2:2:2 to produce Gelation Solution #2.
Vortex.
For reference: Each sample needs ~6 ml of Gelation Solution #2 (~2 ml per incubation round)
Incubate samples in Gelation Solution #2 on ice (3x 10 mins @ 4ºC) with 2ml of Gelation Solution #2 on a shaker in a 12-well plate.
4 °C
00:30:00
30m
Gelation 2
2h 5m
With spatula, transfer gel block containing fish into a Gelation Chamber #2.
Make sure there are no bubbles at the interface between chamber and gel.
If needed, add a small drop of gelation solution below the gel if air bubbles remain.
Add a small drop of gelation solution on top of the gel to minimize chances of air bubbles forming.
Carefully place coverslip on top (Corning #2855-25)
Reagents
Corning® 25x25 mm Square #2 Cover GlassCorningCatalog #2855-25
Slowly pipette fresh Gelation Solution #2 into Gelation Chamber #2 until full. Avoid any air bubbles on either side.
Solution will hold by water tension.
Wipe off any excess solution with a wipe.
The excess solution will prevent oxygen from reaching the gel too quickly, and avoid the inhibition of polymerization.
Note
Avoiding bubbles is critical. If bubbles appear, the procedure should be repeated (cover slip removed and repositioned) until no bubbles are present. Bubbles contain oxygen, which inhibits polymerization by scavenging the free radicals that drive the chain reaction. Moreover, bubbles can become trapped within the gel and cause distortions and artifacts during imaging. Bubbles further than ~1mm away from the sample are acceptable though should still be minimized.
Once Gelation Chamber #2 is filled, place the chambers at 37 ºC for 2 hr to induce polymerization.
Ensure incubator is humidified.
37 °C
02:00:00
2h
Place gels at RT, and allow them to cool on the bench for > 5 min.
Room temperature
00:05:00
5m
Take off coverslip lid with a razor blade.
Under a stereomicroscope, remove the scotch side walls and trim the gels into a rectangle, close to the first gel.
Removing any second gel material that formed outside of the first gel.
Wash in 1x PBS (2x 10 min).
Staining
4h
Incubate gels in Alexa488-NHS and/or Atto647N-maleimide dye at 1:1000 each in 1x PBS (1 hr at RT) in 12-well plate on gentle shaker. DAPI can also be added to label nuclei.
Room temperature
01:00:00
Both Alexa488-NHS and/or Atto647N-maleimide give great contrast and highlight complimentary features of the ultrastructure. Alexa488 is more hydrophilic than Atto647N-maleimide. Total protein dyes give a lot of context to interpret immunohistochemistry results - we recommend starting with Alexa488-NHS.
If staining is not needed, this step can be fully skipped.
Note
Be careful when storing these dyes. A desiccant should be added to container storing the stocks to avoid hydrolysis of the compounds.
Reagents
Alexa Fluor™ 488 NHS Ester (Succinimidyl Ester)Thermo Fisher ScientificCatalog #A20000
ATTO 647N maleimideAAT BioquestCatalog #2857
1h
Wash in 1x PBS (3x 1 hr) on shaker at RT. Washing thoroughly is critical as unbound dye will increase background. Run the washes with a large excess volume. This is key, as long washes in smaller volumes won't result in
Room temperature
03:00:00
3h
Mounting
Attach a 8mm circular coverslips (CS-8R, Warner Instruments, 64-0701) to a Z1 sample holder using superglue.
Reagents
CS-8R Coverslips, 0.15 mm (0.006 in), 8 mm diameter, pkg of 100Multi Channel Systems MCS GmbHCatalog #640701
LIGHTSHEETHOLDER V13 Short.ipt 
LIGHTSHEETHOLDER V13 Short.stl Coat coverslip with poly-lysine and let it dry.
Mount gel, sample side up.
Imaging
Image using a Zeiss Z1 Light sheet Microscope. Allow 45 min (with gel in place) for system to equilibrate before acquiring multi-tile acquisitions.
Image with 20x 1.0 NA water immersion objective.
Exposure time: 100-150 ms.
After imaging, gels can be teased off holder with a paint brush.
For long term storage, keep gels in 1x PBS at 4ºC.
Protocol references
1. Kunst, M. et al. A Cellular-Resolution Atlas of the Larval Zebrafish Brain. Neuron 103, 21-38.e5 (2019).
2. Randlett, O. et al. Whole-brain activity mapping onto a zebrafish brain atlas. Nat Methods 12, 1039–1046 (2015).
3. Tabor, K. M. et al. Brain-wide cellular resolution imaging of Cre transgenic zebrafish lines for functional circuit-mapping.
4. Copper, J. E. et al. Comparative analysis of fixation and embedding techniques for optimized histological preparation of zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 208, 38–46 (2018).
5. Ding, Y. et al. Computational 3D histological phenotyping of whole zebrafish by X-ray histotomography. eLife 8, e44898 (2019).
6. Steib, E. et al. TissUExM enables quantitative ultrastructural analysis in whole vertebrate embryos by expansion microscopy. Cell Reports Methods 2, 100311 (2022).
7. Sim, J. et al. Nanoscale resolution imaging of the whole mouse embryos and larval zebrafish using expansion microscopy. Preprint at https://doi.org/10.1101/2021.05.18.443629 (2021).
8. Chen, F., Tillberg, P. W. & Boyden, E. S. Expansion microscopy.
9. Wang, Y. et al. EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization. Cell 184, 6361-6377.e24 (2021).