May 27, 2026
Version 2
Immunolabeling of a Whole Mouse Brain V.2
- Rajvi Javeri1,
- Laura Roy1,
- Holly Myers1,
- Naveen Ouellette1,
- Molly Logsdon1,
- Andrew Recknagel2,
- Kevin Cao1,
- Judith Baka1,
- Jayaram Chandrashekar1
- 1Allen Institute / Neural Dynamics;
- 2Janelia Research Campus
- Allen Institute for Neural Dynamics
Protocol Citation: Rajvi Javeri, Laura Roy, Holly Myers, Naveen Ouellette, Molly Logsdon, Andrew Recknagel, Kevin Cao, Judith Baka, Jayaram Chandrashekar 2026. Immunolabeling of a Whole Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1okwylr2/v2Version created by Hannah Belski
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: April 01, 2025
Last Modified: June 02, 2026
Protocol Integer ID: 125903
Keywords: immunolabeling, whole brain, antibody, delipidated whole brain sample, fluorescent protein, whole mouse brain, viral labeling of target neuron population, encoded fluorescent protein, whole brain sample, mouse brain, target neuron population, antibody labeling, accessible to antibody labeling, immunolabeling, concentration of fluorophore, fluorophore, organic solvent delipidation, protein, delipidation, signal from endogenous fp
Funders Acknowledgements:
Allen Institute
Abstract
The mouse brains prepared using this protocol have sparse viral labeling of target neuron populations with genetically encoded fluorescent proteins (FPs). Delipidation ensures that whole brain samples become optically clear and more accessible to antibody labeling. However, the signal from endogenous FPs is often dimmed or quenched during organic solvent delipidation. Tissue expansion also dilutes the concentration of fluorophores. Immunolabeling is necessary to amplify signals of FPs for high resolution, multi-scale imaging, of delipidated whole brain samples.
Materials
PBS - Phosphate-Buffered Saline (10X) pH 7.4Invitrogen - Thermo FisherCatalog #AM9625
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Tween 20Catalog #P1379
5% Sodium AzideFisher ScientificCatalog #71448-16
5M NaCl solutionThermo Fisher ScientificCatalog #AM9759
Anti-GFP antibody (ab290)AbcamCatalog #Ab290
Anti-tdTomatoSICGENCatalog #AB8181-200
Donkey anti-Rabbit IgG (H L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488Invitrogen AntibodiesCatalog #A-21206
Donkey anti-Goat IgG (H L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 568 Thermo Fisher ScientificCatalog #A11057
Equipment
VWR® Standard Orbital Shaker, Model 3500
NAME
Shaker
TYPE
VWR
BRAND
89032-092
SKU
LINK
Equipment
Eppendorf™ Centrifuge 5425/5425 R
NAME
Eppendorf™
BRAND
16845110
SKU
LINK
Equipment
WHEATON® Liquid Scintillation Vials, Borosilicate Glass, 22-400 White Urea Caps Attached to Vials, Polyethylene Cone Liner, 20 m
NAME
WHEATON
BRAND
986546
SKU
LINK
Equipment
WHEATON® Shorty Vials clear with PTFE faced rubber lined cap
NAME
vial
TYPE
WHEATON
BRAND
DWKW224607
SKU
LINK
Equipment
Nutating Mixer
NAME
Mixer
TYPE
Fisherbrand
BRAND
88-861-043
SKU
LINK
16.3 x 11.5 x 10.7 in.(415 x 293 x 273 mm)
SPECIFICATIONS
RECIPES
PTxw: 0.05% Tween 20, 0.1% Triton X-100, 0.04% NaN3 in PBS
Combine the following reagents and stir at Room temperature until fully dissolved, store at Room temperature .
| Reagent | Amount | |
| Milli-Q Water | up to 500 mL | |
| 10X PBS | 50 mL | |
| Triton X-100 | 500 uL | |
| Tween 20 | 250 uL | |
| 5% Sodium azide | 4 mL | |
| Total | 500 mL |
Post Secondary Staining PTxw: 0.1% Tween 20, 0.1% Triton X-100, 0.04% NaN3, 200mM NaCl in PBS
Combine the following reagents and stir at Room temperature until fully dissolved, store at Room temperature .
| Reagent | Amount | |
| Milli-Q Water | 864 mL | |
| 10X PBS | 96 mL | |
| Triton X-100 | 1000 uL | |
| Tween 20 | 1000 uL | |
| 5% Sodium Azide | 8 mL | |
| 5M Sodium Chloride Solution | 40 mL | |
| Total | 1000 mL |
Safety warnings
PTxw contains harmful chemicals like 5% Sodium Azide. Use appropriate gloves and PPE while handling, avoid contact with skin or clothing, and dispose of it properly in a hazardous waste stream.
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
If performing the Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy protocol, start with a brain that has been delipidated using the Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain and Aqueous (SBiP) Delipidation of a Whole Mouse Brain protocols. This protocol can also be used to immunolabel brains delipidated using LifeCanvas clearing, iDISCO, or other similar clearing methods.
Applying Primary Antibody
4w 1d 22h
Start with a whole mouse brain perfused with 4% PFA, post-fixed, and stored in PBS in a 20 mL vial.
Using a 20 mL glass vial, wash the brain with PTxw, rotating at Room temperature Overnight .
Transfer the brain to a small 4 mL Shorty Vial containing diluted primary antibody in ~ 4 mL PTxw and incubate with rocking, a nutator, or shaker at 4 °C for 11 days.
- 15 μg/brain for primary antibody
- 30 μg/brain for primary conjugated antibody
1w 4d
Transfer the brain to a 20 mL Liquid Scintillation vial and wash with PTxw, rocking on a nutating mixer or shaker at Room temperature , and replacing solution for each step:
- PTxw for 01:00:00
- PTxw for 02:00:00
- PTxw for 04:00:00
- PTxw Overnight
- PTxw for 24:00:00
- PTxw for 24:00:00
- PTxw for 24:00:00 +
3d 11h
Applying Secondary Antibody
Spin down the desired secondary antibody in a centrifuge set to10.000 rpm for 00:01:00 and use the supernatant for immunolabeling.
Transfer the brain to a small 4 mL Shorty vial containing diluted secondary antibody in ~4 mL PTxw and incubate with rocking at 4 °C for 11 days.
- 30 μg/brain for secondary antibody
Prepare a new solution of PTxw containing 200mM NaCl and 0.1% Tween, and only use this new solution for the remaining washes.
Transfer brain to a 20 mL Liquid Scintillation vial and wash with new PTxw, rocking at Room temperature and replacing solution for each step:
- PTxw for 01:00:00
- PTxw for 02:00:00
- PTxw for 04:00:00
- PTxw Overnight
- PTxw for 24:00:00
- PTxw for 24:00:00
- PTxw for 24:00:00 +
Wash the brain with 1X PBS, rocking at Room temperature Overnight .
Store the immunolabeled brain in 0.01% PBS Azide at 4 °C