Aqueous (SBiP) Delipidation of a Whole Mouse Brain
Naveen Ouellette, Andrew Recknagel, Kevin Cao, Judith Baka, Molly Logsdon, Jayaram Chandrashekar
Abstract
Aqueous strategies for whole-brain delipidation involve lipid removal via phase separation and detergent washes. SBiP is an aqueous biphasic buffer that extracts lipids at polar-nonpolar solvent interfaces. The brain is then washed with the detergent-based B1n buffer, which further disrupts residual membranes, forming micelles that can be washed out. When paired with organic delipidation, these steps can return a solvent-shrunken brain to normal size in phosphate buffer, suitable for post-delipidation antibody labeling.
Before start
If performing this delipidation as part of the Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy protocol, first delipidate the whole mouse brain with organic solvents using the THF and DCM Delipidation of a Whole Mouse Brain protocol.
Steps
SBiP Delipidation
Delipidate with aqueous SBiP solution
Start with a whole mouse brain perfused with 4% PFA, post-fixed, stored in PBS in a 20 mL vial.
Use a 20mL vial for processing an adult mouse brain. All steps in this section are carried out on a carousel rotator 10rpm.
Replace solution in vial with 20mL SBiP for each of the following steps:
- SBiP for
3h 0m 0s - SBiP for
6h 0m 0s - SBiP
- SBiP for
24h 0m 0s - SBiP for
24h 0m 0s - SBiP for
24h 0m 0s - SBiP for
24h 0m 0s
Wash the brain with 20mL B1n buffer at Room temperature .
Replace B1n buffer to complete ~24h 0m 0s wash.
Wash the brain with 1X PBS, rotating at Room temperature for the following steps:
- PBS for
1h 0m 0s - PBS for
2h 0m 0s
Aqueous (SBiP) delipidation complete. Store in 1X PBS 0.05% Azide for up to 6 months.
