Jun 06, 2023

Public workspaceAqueous (SBiP) Delipidation of a Whole Mouse Brain

DOI
dx.doi.org/10.17504/protocols.io.n2bvj81mwgk5/v1
  • 1Janelia Farm;
  • 2Allen Institute for Neural Dynamicss
Naveen Ouellette
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj81mwgk5/v1
Protocol Citation: Andrew Recknagel, Kevin Cao, Naveen Ouellette, Molly Logsdon, Judith Baka, Jayaram Chandrashekar 2023. Aqueous (SBiP) Delipidation of a Whole Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj81mwgk5/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: 79363
Keywords: delipidation, aqueous delipidation, clearing
Funders Acknowledgement:
Allen Institute
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.
Guidelines
It is recommended to etch identification information on the glass vial. Ink or label adhesive may dissolve if exposed to organic solvents.
Materials

ReagentSodium Dodecyl SulfateMerck MilliporeSigma (Sigma-Aldrich)Catalog #74225

ReagentSodium phosphate dibasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #7558-79-4

ReagentSodium phosphate monobasic monohydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9638

Reagent2-methyl-2-butanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #152463

Reagent2-propanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #278475

ReagentGlycineFisher ScientificCatalog #BP381-500

ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Invitrogen - Thermo FisherCatalog #AM9625

ReagentTriton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML

Reagent5% Sodium AzideFisher ScientificCatalog #71448-16

Reagent10N NaOHMerck MilliporeSigma (Sigma-Aldrich)Catalog #SX0607N-6


Equipment
WHEATON® Liquid Scintillation Vials, Caps Attached to Vials, Glass, Polyethylene Cone, 22-400, 20 mL
NAME
Vial
TYPE
Wheaton
BRAND
DWK986546
SKU
https://www.dwk.com/na/wheaton-liquid-scintillation-vials-caps-attached-to-vials-glass-polyethylene-cone-22-400-20-ml-986546
LINK
20 mL Glass Vial with Polyethylene cone Caps
SPECIFICATIONS

Equipment
Fisherbrand™ Multi-Purpose Tube Rotator
NAME
Carousel
TYPE
FisherBrand
BRAND
88-861-049
SKU
https://www.fishersci.com/shop/products/multi-purpose-tube-rotators/88861049
LINK
RECIPES

10 mM Phosphate Buffer pH 8.3

Combine the following reagents, adjust pH to 8.3.
ReagentAmountFinal Concentration
1M Phosphate Buffer5 mL10 mM
Milli-Q water495 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 TemperatureOn ice until solution is uniform and clear. Store immediately at Temperature4 °C until ready for use. Use each batch within a month for best effect.
ReagentVolume
Milli-Q water (ice cold)350 mL
50mM Na2HPO42 mL
4% SDS (in H2O, pH 7.4)10 mL
2-Methyl-2-butanol80 mL
2-Propanol40 mL
Total482 mL

B1n Buffer: 0.1% Triton X-100, 2% Glycine, 0.02% NaN3 in H2O

Combine the following reagents and stir at TemperatureRoom temperature until fully dissolved. Store for a few months atTemperatureRoom temperature .
AB
Milli-Q Waterup to 500 mL
Triton X-100500 uL
Glycine10 g
10N NaOH50 uL
5% Sodium azide2 mL
Total500 mL




Safety warnings
2-Methyl-2-butanol and 2-propanol are corrosive and flammable. Perform the steps that involve these reagents under the fume hood. Wear a lab coat, safety goggles or glasses, and gloves.
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.
SBiP Delipidation
SBiP Delipidation
5d 12h
5d 12h
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 Amount20 mL vial for processing an adult mouse brain. All steps in this section are carried out on a carousel rotator Shaker10 rpm, Room temperature .
Replace solution in vial with Amount20 mL SBiP for each of the following steps:
  • SBiP for Duration03:00:00
  • SBiP for Duration06:00:00
  • SBiP DurationOvernight
  • SBiP for Duration24:00:00
  • SBiP for Duration24:00:00
  • SBiP for Duration24:00:00
  • SBiP for Duration24:00:00


Note
It is important that the SBiP solution is mixed thoroughly during the aqueous steps in order to effectively delipidate the brain. When it is thoroughly mixed, the solution should look uniformly turbid at TemperatureRoom temperature .

Safety information
2-Methyl-2-butanol and 2-propanol are corrosive and flammable. Perform the steps that involve these reagents under the fume hood. Wear a lab coat, safety goggles or glasses, and gloves.


4d 9h
Wash the brain with Amount20 mL B1n buffer at TemperatureRoom temperature DurationOvernight .
Replace B1n buffer to complete ~Duration24:00:00 wash.
1d
Wash the brain with 1X PBS, rotating at TemperatureRoom temperature for the following steps:
  • PBS for Duration01:00:00
  • PBS for Duration02:00:00 +

3h
Aqueous (SBiP) delipidation complete. Store in 1X PBS 0.05% Azide for up to 6 months.