Jun 06, 2023

Public workspaceImmunolabeling of a Whole Mouse Brain

DOI
dx.doi.org/10.17504/protocols.io.ewov1okwylr2/v1
  • 1Janelia Farm;
  • 2Allen Institute for Neural Dynamics
Naveen Ouellette
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DOI: dx.doi.org/10.17504/protocols.io.ewov1okwylr2/v1
Protocol Citation: Andrew Recknagel, Kevin Cao, Molly Logsdon, Judith Baka, Naveen Ouellette, Jayaram Chandrashekar 2023. Immunolabeling of a Whole Mouse Brain. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1okwylr2/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 24, 2023
Last Modified: June 18, 2024
Protocol Integer ID: 79369
Keywords: immunolabeling, whole brain, antibody
Funders Acknowledgement:
Allen Institute
Abstract
Our mice 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 steps. Tissue expansion also dilutes the concentration of fluorophores. To amplify signals of FPs for high resolution, multi-scale imaging, we immunolabel delipidated brain samples.
Materials

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

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

ReagentTween 20Catalog #P1379

Reagent5% Sodium AzideFisher ScientificCatalog #71448-16


Equipment
VWR® Standard Orbital Shaker, Model 3500
NAME
Shaker
TYPE
VWR
BRAND
89032-092
SKU
https://us.vwr.com/store/product/4901055/vwr-standard-orbital-shaker-model-3500
LINK

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
WHEATON® Shorty Vials clear with PTFE faced rubber lined cap
NAME
vial
TYPE
WHEATON
BRAND
DWKW224607
SKU
https://www.sigmaaldrich.com/US/en/product/aldrich/dwkw224607
LINK

Equipment
Nutating Mixer
NAME
Mixer
TYPE
Fisherbrand
BRAND
88-861-043
SKU
https://www.fishersci.com/shop/products/nutating-mixers-variable-speed/88861043
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 TemperatureRoom temperature until fully dissolved, store at Temperature4 °C .
Reagent Amount
Milli-Q Waterup to 500 mL
10X PBS50 mL
Triton X-100500 uL
Tween 20250 uL
5% Sodium azide4 mL
Total500 mL


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.

Immunolabeling of a Whole Mouse Brain
Immunolabeling of a Whole Mouse Brain
4w 3d
4w 3d
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 TemperatureRoom temperature DurationOvernight
Transfer the brain to a small 4 mL vial containing diluted primary antibody in ~ Amount4 mL PTxw and incubate with rocking, a nutator or shaker, at TemperatureRoom temperature for 11 days.
  • 10 μg/brain for primary antibody
  • 20 μg/brain for primary conjugated antibody

1w 4d
Transfer the brain to a 20 mL glass vial and wash with PTxw, rocking on a nutator or shaker at TemperatureRoom temperature , and replacing solution for each step:

  • PTxw for Duration01:00:00
  • PTxw for Duration02:00:00
  • PTxw for Duration04:00:00
  • PTxw DurationOvernight
  • PTxw for Duration24:00:00
  • PTxw for Duration24:00:00
  • PTxw for Duration24:00:00 +

3d 11h
Transfer the brain to a small 4 mL vial containing diluted secondary antibody in ~Amount4 mL PTxw and incubate with rocking at TemperatureRoom temperature for 11 days.
  • 20 μg/brain for secondary antibody
Transfer brain to a 20 mL glass vial and wash with PTxw, rocking at TemperatureRoom temperature , and replacing solution for each step:

  • PTxw for Duration01:00:00
  • PTxw for Duration02:00:00
  • PTxw for Duration04:00:00
  • PTxw DurationOvernight
  • PTxw for Duration24:00:00
  • PTxw for Duration24:00:00
  • PTxw for Duration24:00:00 +
3d 11h
Wash the brain with 1X PBS, rocking at TemperatureRoom temperature DurationOvernight .
16h