Jun 06, 2023
Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain
- Andrew Recknagel1,
- Kevin Cao2,
- Judith Baka2,
- Naveen Ouellette2,
- Molly Logsdon2,
- Jayaram Chandrashekar2
- 1Janelia Farm;
- 2Allen Institute for Neural Dynamics
Protocol Citation: Andrew Recknagel, Kevin Cao, Judith Baka, Naveen Ouellette, Molly Logsdon, Jayaram Chandrashekar 2023. Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj1kxvk5/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: March 23, 2023
Last Modified: June 18, 2024
Protocol Integer ID: 79366
Keywords: delipidation, tissue clearing, clearing
Funders Acknowledgement:
Allen Institute
Abstract
Organic solvent strategies for whole-brain delipidation involve dehydrating the tissue in a water-miscible solvent followed by washes with a water-immiscible solvent. Tissue shrinkage will occur, but when paired an aqueous delipidation step and expansion chemistry becomes negligible. Additionally, an extensive dehydration gradient is used to preserve tissue morphology. We use tetrahydrofuran (THF) as the dehydration solvent, which helps permeabilize cell membranes. THF is then transitioned into dichloromethane as the main delipidation solvent. This protocol produces a delipidated whole brain that can be further processed for expansion or index-matched for immediate imaging.
Guidelines
THF and DCM will damage many types of plastics. Perform delipidation in a glass vial with teflon screw-top cap. Use glass pipets to transfer reagents.
For thorough delipidation, the tissue needs to be dehydrated as much as possible before DCM is added. Use anhydrous tetrahydrofuran (THF) for the 100% THF steps. Store THF and DCM under inert atmosphere. If possible, purchase THF in small volume (100 mL) septa-sealed bottles.
It is recommended to etch identification information on the glass vial. Ink or label adhesive may dissolve if exposed to the solvents used for delipidation.
Materials
DichloromethaneMerck MilliporeSigma (Sigma-Aldrich)Catalog #320269
TetrahydrofuranMerck MilliporeSigma (Sigma-Aldrich)Catalog #186562
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
PYREX® Reusable Serological Pipettes, Glass, Corning, 10 mL
NAME
pipet
TYPE
PYREX®, Corning
BRAND
7085-10
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
Safety warnings
Tetrahydrofuran:
Tetrahydrofuran (THF) is toxic, carcinogenic, and highly flammable. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). Perform the steps that involve these reagents under the fume hood. If these solvents contact your gloves, remove immediately and don new gloves.
When exposed to air, THF may form explosive peroxides if concentrated by distillation or evaporation.
Test for peroxide formation or discard after 1 year.
Dispose of THF in a hazardous waste stream.
Dichloromethane:
Dichloromethane (DCM) is toxic and carcinogenic. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). Perform the steps that involve DCM under the fume hood. If these solvents contact your gloves, remove immediately and don new gloves.
Dispose of DCM in a hazardous waste stream.
Before start
Start with a whole mouse brain perfused with 4% PFA, post-fixed and washed in PBS.
THF and DCM Delipidation of a Whole Mouse Brain
THF and DCM Delipidation of a Whole Mouse Brain
1w
1w
Start with a whole mouse brain perfused with 4% PFA and post-fixed for 03:00:00 to06:00:00 at Room temperature and stored at 4 °C for ~12:00:00 or Overnight , then washed in 1X PBS.
Note
Perform delipidation in a glass vial with teflon screw-top cap. Use glass pipets to transfer reagents.
Note
It is recommended to etch identification information on the glass vial. Ink or label adhesive may dissolve if exposed to organic solvents.
Note
For each step, use ~20 mL or more of solution which should fill to the top of an appropriate sized vial. We use a 20 mL glass scintillation vial.
Dehydrate through H2O → THF series
Wash with tetrahydrofuran (THF) solution (diluted in water) for each whole brain, rotating on a nutator at 4 °C for each step:
- 30% THF for 04:00:00 + or Overnight
- 50% THF for 04:00:00 + or Overnight
- 70% THF for 04:00:00 + or Overnight
- 80% THF for 04:00:00 + or Overnight
- 90% THF for 04:00:00 + or Overnight
- 95% THF for 04:00:00 + or Overnight
Safety information
THF is toxic, carcinogenic, and highly flammable. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). Perform the steps that involve these reagents under the fume hood. If these solvents contact your gloves, remove immediately and don new gloves.
When exposed to air, THF may form explosive peroxides if concentrated by distillation or evaporation.
Test for peroxide formation or discard after 1 year.
Dispose of THF in a hazardous waste stream.
2d
Complete Dehydration Steps
Wash with 100% THF, rotating on a nutator at 4 °C for each step:
- 100% THF for 04:00:00 + or Overnight
- 100% THF Overnight
- 100% THF for 02:00:00 + or Overnight
Note
For thorough delipidation, the tissue needs to be dehydrated as much as possible before DCM is added. Use anhydrous tetrahydrofuran (THF) for the 100% THF steps. Store THF under inert atmosphere. If possible, purchase THF in small volume (100 mL) septa-sealed bottles.
22h
Dichloromethane Delipidation
Delipidate in 100% dichloromethane (DCM), rotating on a nutator at 4 °C for each step:
- 100% DCM for 01:00:00
- 100% DCM for 02:00:00
- 100% DCM for 02:00:00
Note
Store DCM under inert atmosphere.
Safety information
DCM is toxic and carcinogenic. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). Perform the steps that involve DCM under the fume hood. If these solvents contact your gloves, remove immediately and don new gloves.
Dispose of DCM in a hazardous waste stream.
5h
At the end of DCM delipidation, the brain should sink to the bottom of the vial. If not, let the brain incubate longer and perform additional washes until it sinks.
Note
Pause Point:
If the brain is being delipidated using the Whole Mouse Brain Delipidation, Immunolabeling, and ExM protocol, proceed with steps below. If the brain is being delipidated for a DISCO based cleared method, then move forward with appropriate index matching step.
Wash with 100% THF, rotating on a nutator at 4 °C for each step:
- 100% THF for 04:00:00 + or Overnight
- 100% THF Overnight
- 100% THF for 02:00:00 + or Overnight
Safety information
See THF safety information from step 2 above.
22h
Rehydrate through THF → H20 series
Wash with tetrahydrofuran (THF) solution (diluted in water) for each whole brain, rotating on a nutator at 4 °C for each step:
- 95% THF for 02:00:00 + or Overnight
- 90% THF for 02:00:00 + or Overnight
- 80% THF for 02:00:00 + or Overnight
- 70% THF for 02:00:00 + or Overnight
- 50% THF for 02:00:00 + or Overnight
- 30% THF for 02:00:00 + or Overnight
- 10mM Phosphate Buffer pH 8.3 02:00:00 + or Overnight
Safety information
THF is toxic, carcinogenic, and highly flammable. Wear lab coat, safety goggles or glasses, and chemical resistant gloves (7.8 MIL). Perform the steps that involve these reagents under the fume hood. If these solvents contact your gloves, remove immediately and don new gloves.
When exposed to air, THF may form explosive peroxides if concentrated by distillation or evaporation.
Test for peroxide formation or discard after 1 year.
Dispose of THF in a hazardous waste stream.
1d 18h
Wash with 1X PBS, rotating at 4 °C Overnight
Organic solvent (THF/DCM) delipidation complete. Store brains in 1X PBS 0.05% Azide for up to 6 months.