Dec 18, 2025
Whole Mouse Brain Delipidation - LifeCanvas Active
- Holly Myers1,
- Daphne Toglia2
- 1Allen Institute for Neural Dynamics;
- 2Boston University
- Allen Institute for Neural Dynamics
Protocol Citation: Holly Myers, Daphne Toglia 2025. Whole Mouse Brain Delipidation - LifeCanvas Active. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbz5rvx1/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: May 05, 2023
Last Modified: December 19, 2025
Protocol Integer ID: 81474
Keywords: Whole mouse brain delipidation, Brain clearing, Delipidation, whole mouse brain tissue clearing, mouse brains on smartspim, whole mouse brain delipidation, electrophoretic delipidation, whole brain sample, endogenous fluorescence of the tissue, endogenous fluorescence, mouse brain, spill tests on the clearing cup, whole brain imaging, delipidation protocol
Abstract
This LifeCanvas SmartBatch+ protocol is intended for whole mouse brain tissue clearing and incorporates electrophoretic delipidation. While this delipidation protocol is longer than other delipidation protocols we tested, it preserves the endogenous fluorescence of the tissue and clears the whole brain most effectively, demonstrating the morphology of fine cell structures deep in the brain.
It is important to note that while most of our protocol is derived from the original LifeCanvas SmartBatch+ protocol, our protocol has some modifications. Most notably, the SmartBatch+ settings (temperature, time, voltage). While these settings have been found to reliably delipidate whole brain samples, the concern of precipitant build-up and damage to the clearing cup should be acknowledged. Therefore, it is imperative to conduct spill tests on the clearing cup, effectively drain out the conduction buffer, and clean the SmartBatch+ equipment with distilled water after every run.
This delipidation protocol should be followed with a refractive index matching protocol (see: Refractive Index Matching - EasyIndex) and whole brain imaging (see: SmartSPIM setup and alignment and Imaging cleared mouse brains on SmartSPIM).
Materials
Reagents:
LifeCanvas SHIELD Reagent KitCatalog #SH-250
- SHIELD ON Catalog #(SH-ON)
- SHIELD EpoxyCatalog #(SH-Ex)
- SHIELD BufferCatalog #(SH-BS)
Nuclease-free water (e.g. MilliQ or HPLC grade water)
Conduction BufferCatalog #CB
Delipidation BufferCatalog #DB
Optional reagents:
10xPBSAmbionCatalog #AM9624
Sodium Azide 5%Ricca Chemical CompanyCatalog #71448-16
Materials:
| Materials | Product number | |
| Kimwipes | Millipore Sigma, Z188956-60PAK | |
| Aluminum foil | Amazon, B074NB5CDZ | |
| 50ml falcon conical tube | Fisher Scientific, 14-432-22 | |
| Shaker | OHAUS, 404P77 | |
| Metal spatula | Cole Parmer, ux-06287-07 | |
| 50mL serological pipettes | ThermoFisher, 170376N | |
| Serological pipette filler | ThermoFisher, 9501 | |
| Stir plate | MilliporeSigma, Z693510 | |
| Magnetic stirring bar | MilliporeSigma, Z328839 | |
| Pyrex round media storage bottle | MilliporeSigma, CLS13951L | |
| SmartBatch+ accessory kit (includes incubation jar, ring stand, sample ring, mesh bags, lock ring | LifeCanvas, SB-AK |
Equipment
SmartBatch+
NAME
Electrophoretic tissue clearing & immunolabeling chamber
TYPE
LifeCanvas Technologies
BRAND
SB
SKU
LINK
Stochastic electrotransport (SE)
SPECIFICATIONS
Equipment
Clearing Cup
NAME
Clearing Cup for Smartbatch+
TYPE
LifeCanvas Technologies
BRAND
CC
SKU
LINK
SmartBatch+ Accessories
SPECIFICATIONS
Equipment
Mesh Bags
NAME
Mesh Bags to hold whole mouse brains
TYPE
LifeCanvas Technologies
BRAND
MB
SKU
LINK
SmartBatch+ Accessories
SPECIFICATIONS
Equipment
Heratherm General Protocol Microbiological Incubator
NAME
Incubator
TYPE
ThermoFisher
BRAND
51028063
SKU
LINK
75 L
SPECIFICATIONS
Equipment
MaxQ™ HP Incubated Tabletop Orbital Shaker
NAME
MaxQ™ HP, 120 V 60 Hz, 6,5 A o 230 V 50/60 Hz, 3,2 A
TYPE
Thermo Scientific
BRAND
SHKE420HP
SKU
LINK
Recipes:
LifeCanvas SHIELD OFF Solution
Combine the following reagents in the order listed below and stir for 10 minutes. Mix solution over ice or move solution to 4c storage after mixing.
20mL of LifeCanvas SHIELD OFF Solution is required for processing one brain.
| A | B | |
| Nuclease-free water (e.g. MilliQ or HPLC grade water) | 5mL | |
| LifeCanvas SHIELD Buffer | 5mL | |
| LifeCanvas SHIELD Epoxy | 10mL | |
| Total | 20mL |
Optional recipes:
1L 1xPBS:
Combine the following reagents into a container with a stir bar. A graduated cylinder may be used to measure MilliQ water and a graduated cylinder or serological pipet may be used to measure 10xPBS. Mix well on a stir plate at high speed (300 RPM or higher) for 00:02:00 or until solution is mixed. Store at Room temperature for 1 month.
| Reagent | Volume | |
| Milli-Q water | 900mL | |
| 10xPBS | 100mL |
1L 1xPBS & 0.02% Sodium Azide:
Combine the following reagents into a container with a stir bar, using a graduated cylinder to measure the 1xPBS and a P5000 pipette to measure the 5% sodium azide. Mix well on a stir plate at high speed (300 RPM or higher) for 00:02:00 or until solution is mixed. Store at Room temperature or 4 °C for up to 1 year.
| Reagent | Volume | |
| 1xPBS | 996mL | |
| 0.02% Sodium Azide | 4mL |
Troubleshooting
Safety warnings
Make sure to complete all steps with appropriate PPE: gloves, eye protection, lab coat. While it is not known what is in the LifeCanvas reagents for proprietary reasons it is still vital to use PPE when handling all reagents.
Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.
All reagents must be placed in their respective waste bottles and disposed of properly. Please contact Environmental Health & Safety if you need safety labels or more information on properly disposing of all of the LifeCanvas reagents.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
The specimen used for this protocol is a perfused mouse brain fixed in 4% paraformaldehyde. See Mouse Cardiac Perfusion Fixation and Brain Collection V.5 for perfusion and fixation instructions.
Brains should immediately be either stored or index matched when unloaded from SmartBatch+ after delipidation in step 5 of this protocol. If proceeding to index matching, it will be helpful to prepare the 50% EasyIndex solution ahead of time from the protocol Refractive Index Matching - EasyIndex.
Tissue Preservation with SHIELD OFF & SHIELD ON
4d 0h 15m
SHIELD OFF Preservation:
Safety information
Make sure to complete all steps with gloves on, while it is not known what is in these reagents due to proprietary reasons it is still vital to use gloves when handling all reagents.
Prepare 20 mL of fresh SHIELD OFF solution:
Note
The following instructions provide a total volume of 20 mL , which is sufficient to process a single brain specimen. Brains may be processed in batches of 12 or less. If processing more than one brain, 5 mL of nuclease-free water, 5 mL of SHIELD buffer solution, and 10 mL of SHIELD epoxy per brain will be needed, and the amounts of these reagents should be recalculated.
| Reagent | Amount needed | |
| Nuclease-free water (e.g. MilliQ or HPLC grade water) | 5mL | |
| LifeCanvas SHIELD buffer | 5mL | |
| LifeCanvas SHIELD epoxy | 10mL | |
| Total | 20mL |
Place a pyrex storage bottle or similar glass container for mixing the total amount of SHIELD OFF solution needed into a bed of wet ice. Measure the correct amounts of the following reagents in this order into the glass container: nuclease-free water (e.g. MilliQ or HPLC grade water), SHIELD buffer, and SHIELD epoxy. Mix on stir plate for 00:10:00 .
10m
Place each brain into a 50 mL conical tube, add 20 mL of SHIELD OFF, and cover the tube with aluminum foil to protect the tissue from light. Conical tubes should be kept upright in a tube rack throughout this protocol.
Note
The whole mouse brain in the 50 mL conical tube will be completely submerged in the SHIELD OFF solution.
Place 50 mL conical tube in 4 °C fridge with the shaker at a low shaking speed.
Leave the conical tube containing the brain and SHIELD OFF in 4 °C fridge with the shaker for 72:00:00
3d
SHIELD ON Preservation:
Pre-Warm SHIELD ON Buffer by placing in 37 °C oven for 00:05:00 .
5m
Remove the 50 mL conical tubes containing the brain specimen in SHIELD OFF from the 4 °C fridge. Remove SHIELD OFF solution from the conical tube by scooping brain out of tube with a spatula and pouring SHIELD OFF solution into appropriate waste receptacle. Replace the brain in the tube and add 20 mL of SHIELD ON solution into the tube using a serological pipet.
Note
While pipeting reagents into tube containing the brain, point tip of pipet at the inner side wall of tube so that the initial force of the reagent stream hits the tube wall and not the brain.
Place 50 mL conical tubes with 20 mL of SHIELD ON solution in oven at 37 °C with shaking for 24:00:00 . Tubes should be upright inside a tube holder.
1d
SHIELD Preservation is now complete. At this point, the sample can either proceed to Passive Delipidation (step 3) or be stored in20 mL 1X PBS with 0.02% sodium azide at 4 °C for several months without significant loss of fluorescence signal and structural integrity. To store in 1XPBS with 0.02% sodium azide, remove the SHIELD ON solution from the conical tube by scooping brain out of tube with a spatula and pouring SHIELD ON solution into appropriate waste receptacle. Replace the brain in the tube and add 20 mL of 0.02% sodium azide into the tube using a serological pipet.
Safety information
Sodium Azide is toxic and carcinogenic. It should be handled and prepared with care. Do not breathe dust, do not use metal utensils. Wear gloves when handling this chemical.
Passive Delipidation
1d 18h
Passive Delipidation:
Incubate the sample in Delipidation Buffer for24:00:00 at 45 °C on a shaker at slow shaking speed. If you do not have a45 °C incubator, 37 °C will suffice. The step can be extended three extra nights if needed (example: over the weekend), but do not skip it.
If processing a single sample, you may complete the passive delipidation step in the 50mL conical tube. See step 3.1.
If processing multiple samples together as a batch, complete the passive delipidation step using the incubation jar. See step 3.2.
1d
Remove SHIELD ON solution from the conical tube (or 1XPBS & 0.02% sodium azide solution, if the sample was in storage) by scooping brain out of tube with a spatula and pouring SHIELD ON solution into appropriate waste receptacle. Replace the brain in the tube and add 20 mL of Delipidation Buffer into the tube using a serological pipet. The sample is now ready for incubation.
Remove SHIELD ON solution from the conical tube (or 1XPBS & 0.02% sodium azide solution, if the sample was in storage) by scooping brain out of tube with a spatula and pouring SHIELD ON solution into appropriate waste receptacle. Place each mouse brain sample into a lettered mesh bag and hang on metal Ring Stand. When all samples are loaded onto the Ring Stand, lock them in place with the plastic Lock Ring. To track the sample that is in the incubation jar and prevent mix-up between samples, write down the sample ID with the corresponding letter of the mesh bag.
Place the Ring Stand with the samples in the mesh bags into the Incubation Jar. Fill the Incubation Jar with 250 mL of Delipidation Buffer. The samples are now ready for incubation
Example: Mesh bag labeled with the letter "A" (pictured right), corresponds to "A" written on the lid of the conical tube containing the brain specimen, along with the sample ID and date.
Mouse brain samples in lettered mesh bags on metal Ring Stand
Note
Replace Delipidation Buffer in Incubation Jar after every 10 batches.
Active Delipidation
3d 12h
Electrophoretic Delipidation using SmartBatch+ and SmartBatch+ Set Up:
Make sure Auxiliary Power on SmartBatch+ device control panel is turned off.
Before using, wash SmartBatch+ Conduction Chamber by pouring 450 mL Milli-Q water into Chamber and draining through open drainage valve. Repeat 3 times.
Drain out any liquid from the device and ensure the Drainage Valve is closed. Soak up any remaining delipidation buffer from the Delipidation Chamber using a paper towel and dispose of paper towel in appropriate waste.
Pour a whole 450 mL bottle of Conduction Buffer into the Conduction Chamber. (see figure in step 4.2)
Note
Conduction Buffer may be re-used for up to 168 hours of electrophoretic delipidation in SmartBatch+. Retain bottle for storage of recycled Conduction Buffer. (see step 7.1)
Remove the Clearing Cup from its storage container and wash carefully with a gentle stream of Nuclease-free water.
Note
It is important to keep the membrane of the Clearing Cup hydrated at all times.
Clearing Cup is fragile, handle gently for all steps. Do not touch the mesh sides of the Clearing Cup. Instead, hold the cup by the solid plastic top rim or plastic bottom.
Place the Clearing Cup into a beaker of Nuclease-free water for several minutes to finish washing the cup.
Remove the Clearing Cup from the beaker and dump out any water. Carefully use a kimwipe to soak up any remaining water inside.
Insert the pegs on the bottom of the Clearing Cup into the grooves on the Chamber Hex Piece. Twist the Chamber Hex Piece to lock these two pieces together.
Place Clearing Cup (pictured left) on top of Chamber Hex Piece (pictured right).
Place the Clearing Cup into the Delipidation Chamber. (see figure in step 4.3)
Remove the Sample Ring with the lettered mesh bags containing the brain specimens from the Ring Stand in the Delipidation Jar (from step 3) and place it inside the Clearing Cup. All the mesh bags should fit inside the cup and the ends of the three plastic arms protruding from the sample ring should rest on the edges of the cup.
Note
If sample was processed individually in a 50 mL conical tube for Passive Delipidation (step 3), first transfer the sample into a lettered mesh bag and lock the bag into place on the Sample Ring using the Ring lock (see step 3.2 for details), then proceed to step 4.10.
Fill the Clearing Cup with 35 mL of Delipidation Buffer. All brain samples should be completely submerged in Delipidation Buffer.
Turn on Auxiliary Power on control panel.
Close the Chamber Lid and secure it with the thumbscrew.
Close the Case Lid.
Press “Preset” until it indicates Clearing Mode. Change settings to 40V, 1500 mA current limit,42:00:00 , and 42 °C .
1d 18h
Turn on the Electrophoresis Power on control panel.
Mark the time when this active delipidation step started or set a timed shutdown for 42:00:00 on control panel.
1d 18h
Unloading Brain Specimens from SmartBatch+:
Note
Brains should immediately be either stored or index matched when unloaded from SmartBatch+ after delipidation. If proceeding to index matching, it will be helpful to prepare the 50% EasyIndex solution ahead of time from the protocol Refractive Index Matching - EasyIndex.
After electrophoretic delipidation finishes running, open the Case Lid and Chamber Lid. Lift the Sample Ring out of the Clearing Cup and place it on the Ring Stand.
One at a time, remove each brain from its mesh bag hanging on the Ring Stand. Note the letter on the mesh bag and place the brain inside a 50mL conical tube. Label the tube with the corresponding brain specimen ID (see step 3.2).
Immediately store brains (proceed to step 5.3) or immediately begin index matching. Refer to Refractive Index Matching - EasyIndex protocol for instructions on index matching.
To store brains after delipidation, fill each 50 mL tube with 20 mL 1xPBS + Azide 0.02% and place in 4 °C . Brains may be stored for 2-3 months.
SmartBatch+ Clean Up
1w 4d 4h 5m
Unloading Clearing Cup from SmartBatch+:
Remove the Clearing Cup from the Delipidation Chamber and empty the Delipidation Buffer from Clearing Cup into appropriate waste receptacle. Unscrew the Chamber Hex Piece from the Clearing Cup.
Test the Clearing Cup for leakage:
Gently pat the solid plastic base of Clearing Cup dry with a kimwipe and carefully fill with Nuclease-free water. Place Clearing Cup on a paper towel and leave for 00:05:00 .
If the paper towel is dry and there is no evidence of leakage, empty Clearing Cup and replace in storage solution.
If the paper towel is wet, this may indicate Clearing Cup leakage. Repeat this step, and if the paper towel is consistently wet, use a new Clearing Cup next time using SmartBatch+.
5m
Drain Conduction Buffer from SmartBatch+:
Conduction Buffer may be re-used for up to 168:00:00 running in SmartBatch+ electrophoretic delipidation. Drain Conduction Buffer into bottle to retain for extra use if Buffer has been used in SmartBatch+ less than 100:00:00 .
1w 4d 4h
Protocol references