TITLE: Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain
AUTHORS: Andrew Recknagel, Kevin Cao, Judith Baka, Naveen Ouellette, Molly Logsdon, Jayaram Chandrashekar

[DESCRIPTION]
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.

[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.

[STEPS]
SECTION: THF and DCM Delipidation of a Whole Mouse Brain
1. Start with a whole mouse brain perfused with 4% PFA and post-fixed for 180 min  to360 min at Room temperature and stored at 4 °C for ~720 min or 720 min, then washed in 1X PBS.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
 

 

 

SECTION: THF and DCM Delipidation of a Whole Mouse Brain
2. 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 240 min or 240 min 
50% THF for 240 min or 240 min 
70% THF for 240 min or 240 min 
80% THF for 240 min or 240 min 
90% THF for 240 min or 240 min 
95% THF for 240 min or 240 min 

 

SECTION: THF and DCM Delipidation of a Whole Mouse Brain
3. Complete Dehydration Steps
Wash with 100% THF, rotating on a nutator at 4 °C for each step:
100% THF for 240 min or 240 min 
100% THF 240 min 
100% THF for 120 min or 240 min

 

SECTION: THF and DCM Delipidation of a Whole Mouse Brain
4. Dichloromethane Delipidation
Delipidate in 100% dichloromethane (DCM), rotating on a nutator at 4 °C for each step:
100% DCM for 60 min 
100% DCM for 120 min 
100% DCM for 120 min 

 


 


SECTION: THF and DCM Delipidation of a Whole Mouse Brain
5. Wash with 100% THF, rotating on a nutator at 4 °C  for each step:
100% THF for 240 min or 240 min 
100% THF 240 min 
100% THF for 120 min or 240 min


 

SECTION: THF and DCM Delipidation of a Whole Mouse Brain
7. Wash with 1X PBS, rotating at 4 °C   


SECTION: THF and DCM Delipidation of a Whole Mouse Brain
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.
 

SECTION: THF and DCM Delipidation of a Whole Mouse Brain
8. Organic solvent (THF/DCM) delipidation complete. Store brains in 1X PBS 0.05% Azide for up to 6 months.
SECTION: THF and DCM Delipidation of a Whole Mouse Brain
6. 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 120 min or 240 min 
90% THF for 120 min or 240 min 
80% THF for 120 min or 240 min 
70% THF for 120 min or 240 min 
50% THF for 120 min or 240 min 
30% THF for 120 min or 240 min 
10mM Phosphate Buffer pH 8.3  120 min or 240 min