Jun 07, 2023
Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy
- Naveen Ouellette1,
- Andrew Recknagel2,
- Kevin Cao1,
- Judith Baka1,
- Jayaram Chandrashekar1,
- Molly Logsdon1
- 1Allen Institute for Neural Dynamics;
- 2Janelia Farm
- Allen Institute for Neural Dynamics
Protocol Citation: Naveen Ouellette, Andrew Recknagel, Kevin Cao, Judith Baka, Jayaram Chandrashekar, Molly Logsdon 2023. Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldpwjxl5b/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: January 13, 2023
Last Modified: June 19, 2024
Protocol Integer ID: 75235
Keywords: Expansion Microscopy, Whole Brain, Tissue Clearing, Delipidation, Hydrogel, Immunolabeling, Light Sheet, Clearing, Antibody, SPIM, expansion microscopy the mammalian brain, wide single neuron reconstruction, resolution selective plane illumination microscopy, expanded whole mouse brain, selective plane illumination microscopy, contrast imaging of entire brain, isotropic expansion of whole mouse brain, expansion microscopy, whole mouse brain delipidation, whole mouse brain, whole brain data set, mammalian brain, spim microscope without need, complete axonal morphology of individual neuron, spim microscope, neuronal axon, entire brain, description of the brain, individual neuron, imaging, brain area, brain region, wide neural circuit, obtaining brain, tracing complete axonal morphology, neuron, individual axon collateral, contrast imaging, brain, wide neural circuits during normal behavior, distinct neuron type, resolution
Funders Acknowledgements:
Allen Institute
Abstract
The mammalian brain contains approximately 1,000 brain areas and each brain area contains multiple (up to 100) cell types. Neurons in one brain region can send projections to dozens of target regions, and distinct neuron types could project to different combinations of target regions. The enumeration and description of the brain’s cell types, and their brain-wide connectivity, is foundational for understanding how neural activity is routed across brain-wide neural circuits during normal behavior and how these processes are dysregulated in mental disorders.
Obtaining brain-wide single neuron reconstructions requires high-resolution, high-contrast imaging of entire brains – neuronal axons travel many centimeters (in the mouse) while individual axon collaterals could be finer than 100nm. Here, we present an integrated protocol for labeling and isotropic expansion of whole mouse brains that results in optically clear specimens ideally suited for high-resolution selective plane illumination microscopy (SPIM) imaging. Pipeline steps are modular, and the protocol is extensible to other large volume clearing and expansion applications.
We have imaged expanded whole mouse brains generated using this protocol on our custom built ExA-SPIM microscope without need for any tissue slicing. These whole brain data sets are being used for tracing complete axonal morphology of individual neurons.
Materials
DichloromethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #320269
TetrahydrofuranMerck MilliporeSigma (Sigma-Aldrich)Catalog #186562
Sodium Dodecyl SulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #74225
Sodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #7558-79-4
Sodium phosphate monobasic monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9638
2-methyl-2-butanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #152463
2-propanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #278475
GlycineFisher ScientificCatalog #BP381-500
PBS - Phosphate-Buffered Saline (10X) pH 7.4Invitrogen - Thermo FisherCatalog #AM9625
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Tween 20Catalog #P1379
5% Sodium AzideFisher ScientificCatalog #71448-16
MES or 2-(N-Morpholino)ethanesulfonic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3671
5M Sodium Chloride, 1000mlPromegaCatalog #V4221
10N NaOHMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0607N-6
Acryloyl-X, SEThermo Fisher ScientificCatalog #A20770
DMSO anhydrousThermo Fisher ScientificCatalog #D12345
AcrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #A9099
NN Methylene-Bis-acrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M7279
Sodium Acrylate (purity note:*) Merck MilliporeSigma (Sigma-Aldrich)Catalog #408220
Acrylic AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #147230
Proteinase K, Molecular Biology Grade - 2 mlNew England BiolabsCatalog #P8107S
1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Phosphate Buffer Solution 1.0 M pH 7.4 (25 °C)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P3619
SSC (20X) RNase-freeInvitrogen - Thermo FisherCatalog #AM9765
EDTA (0.5 M), pH 8.0Life TechnologiesCatalog #AM9260G
Tris-HCl (1M pH 8)Thermo Fisher ScientificCatalog #AM9856
Agarose for ≧1kbp fragmentNacalai Tesque Inc.Catalog #01163-76
| Materials | Product Number | |
| 1 gallon Slider plastic storage bags | Amazon, COMINHKG109462 | |
| Cover glass, 24X55MM | Epredia, 12455S | |
| Diamond Scriber | Ted Pella, 62107-ST | |
| Glass Serological Pipet | Fisher Scientific, PYREX™708710 | |
| Glass Slides, 1"x3" | EMS, 71867-01 | |
| Heavy Duty Carbon Steel Razor | EMS, 71965 | |
| Instrument Soaking Tray | Sklar, 10-3052 | |
| Large Cover Glass, #2 | Brain Research Laboratories, 4342-2 | |
| Pelco Single edge uncoated carbon steel | Ted Pella, 121-95 | |
| S/S or S/A Press to Seal Gasket 32X19mm(D), various depths: 0.5, 1.0, 2.0, 2.5 mm | EMS, 70337 | |
| WHEATON® Shorty Vials clear with PTFE faced rubber lined cap | DWK, DWK986546 | |
| WHEATON® Liquid Scintillation Vials, Caps Attached to Vials, Glass, Polyethylene Cone, 22-400, 20 mL | DWK, DWKW224607 |
Equipment
Hefty Slider Freezer Storage Bags, Gallon Size, 56 Count
NAME
Hefty
BRAND
COMINHKG109462
SKU
LINK
1 gallon
SPECIFICATIONS
Equipment
Cover glass
NAME
Epredia
BRAND
12-455-S
SKU
LINK
24x55 mm
SPECIFICATIONS
Equipment
Deluxe Diamond Scribing Pen
NAME
EMS
BRAND
54468
SKU
LINK
Equipment
PYREX® Reusable Serological Pipettes, Glass, Corning, 10 mL
NAME
pipet
TYPE
PYREX®, Corning
BRAND
7085-10
SKU
LINK
Equipment
Microscope Slides
NAME
slides
TYPE
EMS
BRAND
71867-01
SKU
LINK
25x75mm, thickness: 1mm, Frosted End
SPECIFICATIONS
Equipment
Single Edge Carbon Steel Razor
NAME
blade
TYPE
EMS
BRAND
71960
SKU
LINK
Equipment
Instrument Soaking Tray
NAME
Sklar
BRAND
10-3052
SKU
LINK
Equipment
Large Cover Glass no.2 thickness (0.19 – 0.25mm)
NAME
cover glass
TYPE
Brain Research Laboratories
BRAND
4342-2
SKU
LINK
3-3/4″ x 4-1/2″ (95mm x 114mm) no.2 thickness (50pc/pack)
SPECIFICATIONS
Equipment
Single edge uncoated carbon steel blade
NAME
blade
TYPE
Pelco
BRAND
121-95
SKU
LINK
118mm long x 19mm wide x 0.229mm thick. (4.65 x 0.75 x 0.009")
SPECIFICATIONS
Equipment
S/S Press to Seal Gasket 32X19mm(D)
NAME
EMS
BRAND
70337
SKU
LINK
0.5, 1.0, 2.0, 2.5 mm depths. S/S or S/A
SPECIFICATIONS
Equipment
WHEATON® Liquid Scintillation Vials, Caps Attached to Vials, Glass, Polyethylene Cone, 22-400, 20 mL
NAME
Vial
TYPE
Wheaton
BRAND
DWK986546
SKU
LINK
20 mL Glass Vial with Polyethylene cone Caps
SPECIFICATIONS
Equipment
WHEATON® Shorty Vials clear with PTFE faced rubber lined cap
NAME
vial
TYPE
WHEATON
BRAND
DWKW224607
SKU
LINK
Equipment
Nutating Mixer
NAME
Mixer
TYPE
Fisherbrand
BRAND
88-861-043
SKU
LINK
16.3 x 11.5 x 10.7 in.(415 x 293 x 273 mm)
SPECIFICATIONS
Equipment
Fisherbrand™ Multi-Purpose Tube Rotator
NAME
Carousel
TYPE
FisherBrand
BRAND
88-861-049
SKU
LINK
RECIPES
10 mM Phosphate Buffer pH 8.3
Combine the following reagents, adjust pH to 8.3.
| Reagent | Amount | Final Concentration | |
| 1M Phosphate Buffer | 5 mL | 10 mM | |
| Milli-Q water | 495 mL |
SBiP Solution: 0.08% SDS, 16% 2-methyl-2-butanol, 8% 2-propanol, in H2O
Combine the following reagents on ice. Use a fume hood when adding 2-methyl-2-butanol and 2-propanol. Mix On ice until solution is uniform and clear. Store immediately at 4 °C until ready for use. Use each batch within a month for best effect.
| Reagent | Amount | Final Concentration | |
| 4% SDS (in H2O, pH 7.4) | 10 mL | 0.08% | |
| 2-Methyl-2-butanol | 80 mL | 16% | |
| 2-Propanol | 40 mL | 8% | |
| 50mM Na2HPO4 | 2 mL | ||
| Milli-Q water (ice cold) | 350 mL | ||
| Total | 482 mL |
B1n Buffer: 0.1% Triton X-100, 2% Glycine, 0.02% NaN3 in H2O
Combine the following reagents and stir at Room temperature until fully dissolved. Store for a few months atRoom temperature .
| Reagent | Amount | Final Concentration | |
| Triton X-100 | 500 uL | 0.1% | |
| Glycine | 10 g | 2% | |
| 5% Sodium azide | 2 mL | 0.02% | |
| 10N NaOH | 50 uL | ||
| Milli-Q Water | up to 500 mL | ||
| Total | 500 mL |
PTxw: 0.05% Tween 20, 0.1% Triton X-100, 0.04% NaN3 in PBS
Combine the following reagents and stir at Room temperature until fully dissolved, store at 4 °C .
| Reagent | Amount | Final Concentration | |
| 10X PBS | 50 mL | 1X | |
| Triton X-100 | 500 uL | 0.1% | |
| Tween 20 | 250 uL | 0.05% | |
| 5% Sodium azide | 4 mL | 0.04% | |
| Milli-Q Water | up to 500 mL | ||
| Total | 500 mL |
MBS solution: 100mM MES Buffered Saline, 150mM NaCl, pH 6.0
Combine the following reagents at Room temperature until fully dissolved. Adjust to pH 6. Store for a few months atRoom temperature .
| Reagent | Volume | Final Concentration | |
| MES | 1 g | 100 mM | |
| 5M NaCl | 1.5 mL | 150 mM | |
| 10N NaOH | 200 uL | ||
| Milli-Q Water | 48 mL | ||
| Total | 50 mL |
10 mg/mL Acryloyl-X (AcX) in DMSO:
To make 10 mg/mL AcX, add DMSO directly to the bottle of AcX. Vortex to dissolve. Aliquot and store at -20 °C in a desiccated environment. Aliquots should be used within one month.
| Reagent | Volume | Final Concentration | |
| Acryloyl X | 5 mg | 10 mg/mL | |
| Dimethyl Sulfoxide (DMSO) | 500 uL |
10% (w/v) VA-044 (polymerization initiator):
Combine the following reagents and stir On ice until dissolved. Store at -20 °C up to one month.
| Reagents | Volume | Final Concentration | |
| VA-044 | 1 g | 10% | |
| Milli-Q water | 10 mL |
Stock X (Monomer Solution) Preparation:
To make the Stock X solution, the following stock solutions must be prepared in advance:
- 50% (w/v) Acrylamide
- 2% (w/v) N,N Methylene-bis-acrylamide
- 4.04M Sodium acrylate (2 options: made from acrylic acid or powder form)
50% (w/v) Acrylamide:
Combine the following reagents and stir until dissolved. Store at -20 °C up to one month.
| Reagents | Volume | Final Concentration | |
| Acrylamide | 5 g | 50% | |
| Milli-Q water | 10 mL |
2% (w/v) N,N Methylene-Bis-Acrylamide
Combine the following reagents and stir until dissolved. Store at -20 °C up to one month.
| Reagents | Amount | Final Concentration | |
| N,N Methylene-bis-acrylamide | 0.2 g | 2% | |
| Milli-Q water | 10 mL |
4.04M Sodium Acrylate
Add 22.5 mL milli-Q water into a 250 mL glass bottle and cool the solution down to 0 °C on ice. Slowly add in 27.5 mL acrylic acid with stirring until fully mixed. Cover the bottle to the neck with ice and then, with stirring, add 36 mL 10N NaOH over the course of 00:10:00 . Make sure to keep the solution at 0 °C .
Insert a pH meter and begin adding 1N NaOH in 1 mL increments until pH 7.6 – 8.0. Keep track of the volume needed to reach this range.
Once desired pH is reached, let the solution warm to Room temperature and check the pH to make sure it is still in correct range. Add water to a final volume of 100mL and store at -20 °C .
| Reagents | Amount | |
| 14.6M Acrylic acid | 27.5 mL | |
| Milli-Q water | 22.5 mL + extra to reach 100mL | |
| 10N NaOH | 36 mL | |
| 1N NaOH | ~5-10 mL |
4.04M Sodium Acrylate (from powder form)
Combine the following reagents and stir until dissolved. Store at -20 °C up to one month.
| Reagents | Amount | Final Concentration | |
| Sodium acrylate | 18.99 g | 4.04 M or 38% | |
| Milli-Q water | 50 mL |
Note
Powder Sodium Acrylate can be used. However, a yellow solution indicates low purity. If this is observed, discard and use a different batch.
Stock X (Monomer Solution)
Combine the following On ice . Aliquot and store at -20 °C for up to one month.
| Reagents | Amount | Final Concentration | |
| 4.04M Sodium acrylate | 4.554 mL | 9.2% | |
| 50% Acrylamide | 1 mL | 2.7% | |
| 2% N,N Methylene-bis-acrylamide | 1.5 mL | 0.16% | |
| 5M NaCl | 8 mL | 12% | |
| 10X PBS | 2 mL | 1X | |
| Milli-Q water | 1.745 mL | ||
| Total | 18.799 mL |
Proteinase K Digestion Buffer: 50mM Tris-HCl pH 8, 1mM EDTA, 0.5% TritonX, 50mM NaCl, 0.3%SDS
Combine the following reagents. Aliquot and store at -20 °C for several months.
| Reagent | Amount | Final Concentration | |
| 1M Tris-HCl pH 8 | 2 mL | 50 mM | |
| 10% Triton X-100 | 2.5 mL | 0.5% | |
| 5M NaCl | 500 uL | 50 mM | |
| 0.5M EDTA | 100 uL | 1 mM | |
| 10% SDS | 1.5 mL | 0.3% | |
| Milli-Q Water | 42.9 mL | ||
| Total | 50 mL |
Troubleshooting
Safety warnings
Tetrahydrofuran (THF) and dichloromethane (DCM) are toxic and carcinogenic. THF is flammable. When exposed to air, THF may form explosive peroxides if concentrated by distillation or evaporation. Test for peroxide formation or discard THF after 1 year. Perform the steps that involve these reagents under the fume hood. Dispose of THF and DCM in a hazardous waste stream. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). If these solvents contact your gloves, remove immediately and don new gloves.
2-methyl-2-butanol and 2-propanol are corrosive and flammable. Perform the steps that involve these reagents under the fume hood. Dispose of 2-methyl-2-butanol and 2-propanol in a hazardous waste stream. Wear a lab coat, safety goggles or glasses, and gloves.
Sodium azide may be harmful if inhaled. It may cause respiratory tract, skin, and eye irritation and may be fatal if absorbed through skin or swallowed. Sodium azide can react with metal spatulas and metal lab equipment to form shock sensitive salts. Sodium azide reacts with lead, copper, silver, gold and metal halides to form heavy metal azides which are shock sensitive and explosive. Additionally, contact with acids liberates toxic gas. Dispose of sodium azide in a hazardous waste stream. Wear a lab coat, safety goggles or glasses, and gloves.
Acrylamide powders and solutions are toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, teratogen and a carcinogen. Dispose of acrylamide and any contaminated consumables in a hazardous waste stream. Wear a lab coat, safety goggles or glasses, and gloves.
Protocol Overview
This protocol prepares a whole mouse brain for expansion microscopy (ExM). Methods of tissue processing include organic and aqueous delipidation, immunolabeling, ExM (gel embedding and expansion), and mounting the sample in the imaging chamber.
Tetrahydrofuran / Dichloromethane Delipidation
1w 1d
Reference Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain protocol.
Protocol
CREATED BY
Naveen Ouellette
SBiP Delipidation
1w
Reference Aqueous (SBiP) Delipidation for a Whole Mouse Brain protocol.
Immunolabeling
4w 3d
Reference Immunolabeling of a Whole Mouse Brain protocol.
Gelation and Digestion
4w 2d 6h 23m
Day 1 – MBS equilibration
For all ExM steps, use small-volume glass tubes or vials to contain the brain. This way, we use a smaller amount of valuable reagents for gelation. We typically use a 4 mL glass vial that has a wide enough opening to fit an adult mouse brain.
Wash sample in MBS at Room temperature , filling vial to the top (typically 4 mL ). Replace solution for each step:
- MBS for 01:00:00 +
- MBS for 01:00:00 +
2h
Replace MBS and store On ice at 4 °C Overnight
16h
Day 2-5 – Acryloyl X (AcX) treatment
4d
Prepare a new tube or vial with 3 mL MBS for each brain. Place On ice .
Mix AcX with MBS (2500 µg per brain or 250 µL of 10 mg/mL AcX with 3 mL MBS) for each whole brain.
Replace MBS solution from previous step with AcX solution for each whole brain and fill remaining space in vial with MBS to minimize air.
Incubate On ice at 4 °C , mix by inverting tubes once per day for 4 days.
4d
Day 6-7 – PBS washes
2d
Replace solution with cold 1X PBS, mix by inverting, keep on On ice at 4 °C , Overnight .
16h
Replace solution with cold 1X PBS, mix by inverting, keep on On ice at 4 °C , Overnight .
16h
Day 8-11 – Stock X equilibration
4d
Prepare Stock X on On ice .
Safety information
Acrylamide powders and solutions are toxic if swallowed, inhaled, or absorbed through the skin. It is a mutagen, teratogen and a carcinogen.
Dispose of acrylamide and any contaminated consumables in a hazardous waste stream.
To activate Stock X, add VA-044. The amount of VA-044 required is equal to 1.2% of the total volume of Stock X used. (Typically we add 234 µL of 10% (w/v) VA-044 in ~20 mL Stock X).
Fill each whole brain tube with activated Stock X solution to minimize air.
Save any extra activated Stock X solution on On ice .
Incubate on On ice at 4 °C , mix by inverting tubes once per day for 4 days.
4d
Day 12 – Gelation and Proteinase K
Bring brain vials and activated Stock X solution to Room temperature on the bench. Swirl Stock X conical tube carefully to avoid bubbles.
Note
Typically a 5 mL preparation of activated Stock X is more than sufficient for gelling one adult mouse brain. If more activated Stock X is required, a fresh preparation may be added to the Stock X left over from the previous step.
De-gas activated Stock X solution in a vacuum chamber ~00:20:00 . This reduces the appearance of bubbles during polymerization.
20m
Prepare polymerization chamber by stacking silicone isolator gaskets of various thicknesses to an appropriate height on an uncharged 1"x3" microscope slide. The gaskets should be stacked high enough so the entire mouse brain will be embedded within the hydrogel. Typically, we stack the gaskets to allow about 2 mm of gel to polymerize around the brain. Inspect chamber for dust and debris before beginning embedding process.
Add activated Stock X to partly fill polymerization chamber.
Avoid and eliminate all bubbles.
Transfer brain to chamber and fill with Stock X just to the top.
Seal chamber with a clean, long coverslip, taking care to avoid bubbles.
Place in petri dish, and seal in 2 sequential zip lock bags, each thoroughly purged with nitrogen gas.
Incubate at 37 °C for 04:00:00 +
4h
Remove coverslip and gasket; gel should be firm without extra liquid dripping out.
Cut gel with a razor blade, making a rectangular cuboid shape. Leave about a 2 mm border of gel on all sides of the brain. If needed, the gel may be trimmed down further after expansion.
Wash in 50 mL 1X PBS at Room temperature in a 50 mL conical tube for about 00:03:00 with swirling.
3m
Replace solution with 40 mL Proteinase K (ProK) buffer spiked with 100U ProK (~126 µL ); incubate at Room temperature with gentle swirling or rocking on a nutator for 5 days.
Note
Store Proteinase K at -20 °C and keep On ice until added to the ProK Buffer.
5d
Day 18 – Proteinase K digest (continued)
5d
Swirl whole brain conical tube carefully and thoroughly.
Add 126 µL (100U) Proteinase K to the tube.
Move to 37 °C and incubate 5 days+. Cortex should look transparent and the white matter should look mostly clear. Digestion time may be extended if needed.
Day 23 – Final washes (after judging that digest is complete)
3d
Wash brain in ~50 mL 1X PBS briefly at Room temperature , then replace with enough 1X PBS to fill the 50 mL conical tube; wash at Room temperature with gentle swirling or rocking for 2 days.
2d
Replace 1X PBS solution for a few hours and/or Overnight .
16h
Change final wash with 1X PBS Azide 0.05% and store at 4 °C until ready for expansion.
Expansion of Hydrogel Embedded Brain
Submerge the hydrogel in 0.05X SSC.
- Replace 0.05X SSC once per day for at least 3 days at Room temperature .
The hydrogel should expand to about 3 times its original size before digestion. If the gel does not seem to be expanded fully, change the SSC buffer again.
As it expands, the hydrogel becomes more fragile. When handling the hydrogel, use a gloved hand to carefully transfer it to another container if needed. To exchange 0.05X SSC, we use a 2 L Instrument Soaking Tray which has a strainer insert. The gel and strainer are lifted out of the solution, the solution is refreshed, and the strainer and gel are gently placed back in.
Mounting of Hydrogel in ExA-SPIM Chamber
Mounting of Expanded Hydrogel Embedded Brain in ExA-SPIM Chamber
The expanded hydrogel is embedded in a chamber that will hold it in place during imaging on the ExA-SPIM. The hydrogel is placed against the upper corner of the chamber and held in place with agarose. Once the agarose is solidified, the solid top and front panels that the sample is resting against are replaced with glass. The sample will be securely held in place while allowing access on two sides for imaging.
Assembled ExA-SPIM sample chamber with expanded brain sample and agarose support.
Assemble the glass window panels
Using a glass cutting pen and ruler, cut #2 glass panels to fit the top and front window frames. Along the longest edge, trim the glass about 1 mm shorter than measured. When the panels are assembled on the chamber, this will leave a gap at the corner where the two glass edges meet along the top-front corner. This opening allows fluid to move between the chamber and surrounding imaging buffer, helping keep the sample equilibrated.
Use Krazy glue to apply the glass to the metal frame. Make sure the glass is clean and free of debris or glue.
Optional: a glass window may be cut for the back panel viewing window, but this is not crucial. The back solid panel may remain in place during imaging.
Top and Front glass panels assembled.
Assemble the chamber to prepare for agarose embedding
The chamber is first assembled with solid sides so that the hydrogel can be placed inside and embedded in 2% Agarose without leakage. Leave one side open (Back or Bottom side) for inserting the hydrogel. Fill chamber with water to test for any leaks. If leaks are observed, ensure that all screws are tight. If there is a very slow leak, this is usually OK, agarose will not leak as easily as water.
The chamber is assembled with bottom panel left open for hydrogel insertion.
Trim the expanded hydrogel
The lateral side of the embedded brain hydrogel will be mounted against the front glass panel. This side of the gel should have a smooth surface. If the cut sides of the hydrogel are uneven, it will be difficult to mount without agarose leaking around the uneven side and obscuring the brain.
Use a long, thin blade to smoothly trim the sides along the lateral, anterior and posterior sides of the hydrogel. It is best to leave about 6 mm buffer of empty hydrogel surrounding the brain.
Trimming is not necessary unless the the gel is too large or front facing side is very uneven.
Trimmed hydrogel, smooth cuts on lateral sides.
Position the hydrogel in the imaging chamber
Submerge the hydrogel and assembled chamber in a wide dish filled with the same 0.05X SSC that the brain was soaking in. Position the hydrogel so the dorsoventral axis of the brain intersects with the top and bottom of the chamber (Z-axis of the ExA-SPIM). The dorsal surface should be facing the top panel.
While submerged, carefully use your hand to slide the hydrogel through the open panel into the chamber. Position the chamber so the open panel faces up. The hydrogel should rest against the corner of the Front and Top sides. Carefully drain any 0.05X SSC that is left in the chamber. A transfer pipet may be used to remove any remaining 0.05X SSC.
ExA-SPIM chamber positioned with hydrogel resting in top-right corner.
Prepare 150 mL of 2% agarose. Add 3 g of agarose to 150 mL 0.05X SSC. Stir with a spatula and heat about 2 minutes in the microwave until boiling and solution is clear.
Note
IMPORTANT NOTE: The agarose must be made from the very same batch of 0.05X SSC that the hydrogel was expanding in. Any difference in salt concentration in the agarose can alter the size of the hydrogel after it has been embedded.
Wait until the agarose has cooled to about 55 °C .
To prevent the agarose from leaking underneath the hydrogel, prop the chamber at an angle (against the side of a petri dish) so gravity is gently pulling the hydrogel against the Front-Top corner of the chamber. Gently pour the agarose in so it just reaches the lowest edge of the open panel. Wait for it to solidify. Keep remaining agarose on a hot plate at ~55 °C .
Molten agarose is poured into the chamber.
Once the first pour is solid, place the chamber on a level surface and fill the remaining space with agarose. Insert the last panel and wait for it to solidify. The bottom panel has 4 screw holes that are used to attach it to the imaging chamber. It must placed so the the holes are positioned farther away from the sample.
The front and top panels must be replaced with glass panels to conduct imaging. Carefully remove these panels and replace with the glass panels that were prepared earlier. Avoid trapping bubbles.
It helps to wet the surfaces of the exposed agarose and hydrogel with 0.05X SSC when the new panels are applied. There should be a small gap visible at the top-front corner where the glass panels meet. This opening helps keep the sample equilibrated with the surrounding imaging buffer.
Front panel is removed.
Front and Top panels have been replaced with glass. A small gap remains in the corner to allow for fluid exchange.
Submerge embedded sample back in to the 0.05X SSC it was previously soaking in. Protect container from light and leave the embedded hydrogel to soak overnight.
Mounted sample is submerged in 0.05x SSC before imaging.
The sample is now ready to be imaged on the ExA-SPIM.