Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain

Naveen Ouellette, Andrew Recknagel, Kevin Cao, Judith Baka, Molly Logsdon, Jayaram Chandrashekar

Published: 2023-06-07 DOI: 10.17504/protocols.io.36wgqj1kxvk5/v1

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.

Before start

Start with a whole mouse brain perfused with 4% PFA, post-fixed and washed in PBS.

Steps

THF and DCM Delipidation of a Whole Mouse Brain

Start with a whole mouse brain perfused with 4% PFA and post-fixed for 3h 0m 0s to6h 0m 0s at Room temperature and stored at 4°C for ~12h 0m 0s or 12h 0m 0s, 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 ~20mL 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 ₂O → 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 4h 0m 0s or 4h 0m 0s
50% THF for 4h 0m 0s or 4h 0m 0s
70% THF for 4h 0m 0s or 4h 0m 0s
80% THF for 4h 0m 0s or 4h 0m 0s
90% THF for 4h 0m 0s or 4h 0m 0s
95% THF for 4h 0m 0s or 4h 0m 0s

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.

Complete Dehydration Steps

Wash with 100% THF, rotating on a nutator at 4°C for each step:

100% THF for 4h 0m 0s or 4h 0m 0s
100% THF 4h 0m 0s
100% THF for 2h 0m 0s or 4h 0m 0s

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.

Dichloromethane Delipidation

Delipidate in 100% dichloromethane (DCM), rotating on a nutator at 4°C for each step:

100% DCM for 1h 0m 0s
100% DCM for 2h 0m 0s
100% DCM for 2h 0m 0s

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.

4.1.

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 4h 0m 0s or 4h 0m 0s
100% THF 4h 0m 0s
100% THF for 2h 0m 0s or 4h 0m 0s

Safety information

See THF safety information from step 2 above.

Rehydrate through THF → H20 series ₂0 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 2h 0m 0s or 4h 0m 0s
90% THF for 2h 0m 0s or 4h 0m 0s
80% THF for 2h 0m 0s or 4h 0m 0s
70% THF for 2h 0m 0s or 4h 0m 0s
50% THF for 2h 0m 0s or 4h 0m 0s
30% THF for 2h 0m 0s or 4h 0m 0s
10mM Phosphate Buffer pH 8.3 2h 0m 0s or 4h 0m 0s

Safety information

Wash with 1X PBS, rotating at 4°C

Organic solvent (THF/DCM) delipidation complete. Store brains in 1X PBS 0.05% Azide for up to 6 months.

Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain

Abstract

Before start

Steps

THF and DCM Delipidation of a Whole Mouse Brain

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