Feb 24, 2020
'Uniclear' water-based brain clearing for light sheet imaging of electrode tracks
- 1Janelia Research Campus
- Optical Clearing of Tissue
Protocol Citation: Susu Chen, Karel Svoboda 2020. 'Uniclear' water-based brain clearing for light sheet imaging of electrode tracks. protocols.io https://dx.doi.org/10.17504/protocols.io.zndf5a6
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 31, 2019
Last Modified: February 24, 2020
Protocol Integer ID: 21925
Abstract
UniClear procedure for whole mouse brain clearing and refractive index matching. The advantages of this method are that the method is water-based and produces a mechanically robust specimen. Cleared brains can be imaged with lightsheet microscopes (e.g. Zeiss Z1 lightsheet).
Materials
MATERIALS
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
OptiPrep™ Density Gradient MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1556)
2-Propanol (99.5 %)Merck MilliporeSigma (Sigma-Aldrich)Catalog #278475
2-methyl-2-butanol
2-propanol
Sodium dodecyl sulfate solution (SDS), 10% in H20
OptiPrep™Density Gradient Medium
Dimethyl sulfoxide (DMSO)
Nycodenz
Preparation of SBiP buffer: 500ml
*** Mix SBiP at 4C till fully dissolved, kept at 4C.
At room temperature, SBiP will get activated and emulsified for delipidation. Use each batch within a month for best effect.
- Ice cold H2O 350 ml
- 50mM Na2HPO4 2 ml
- 4% SDS (in H2O, pH7.4) 10 ml
- 2-methyl-2-butanol 80 ml
- 2-propanol 40 ml
Preparation of Opti-prep refractive index matching solution
- 60% (w%) Opti-prep solution
- 20% (w%) DMSO
- 20% (w%) PBS
- 20 mM Tris
- add overall 1.12g/ml Nycodenz (dissolve gradually by adding ~1/10th of Nycodenz powder at a time until fully mixed, heating up and stiring can help to dissolve faster)
References:
Economo, Clark, et al., A plat form for brain-wide imaging and reconstruction of individual neurons.
Winnubst et al., Reconstruction of 1,000 Projection Neurons Reveals New Cell Types and Organization of Long-Range Connectivity in the Mouse Brain
Cell.2019 Sep 19;179(1):268-281.e13. doi: 10.1016/j.cell.2019.07.042. Epub 2019 Sep 5.
Perfuse mouse with cold 4% PFA. Post fix brain for at least 24:00:00 at room temperature (RT). Wash brain in PBS to remove fixative. Fixed brain samples can be kept in PBS at 4 °C before further processing.
Delipidate brain with SBiP buffer: 20 mL per adult mouse brain, shaking (70 RPM) at 37 °C . On the first day: change buffer at 03:00:00 , 06:00:00 , 12:00:00 and leave over night. Starting on the second day: change SBiP buffer every 24:00:00 for consecutive 14 days , shaking (70 RPM) at 37 °C .
After delipidation, wash brain in PBS for at least 12:00:00 before further processing. Sample can be stored in PBS at RT before refractive index matching.
Refractive index matching: first day in 20 mL 50% PBS + 50% opti-prep mixed solution, rotating at RT for 24:00:00 . On second day transfer the brain to 40 mL 100% opti-prep solution, rotating at RT for 24:00:00 . Index-matched brain should appear transparent.
For light-sheet imaging (e.g. Zeiss Z1) mount brain vertically.
Example of a mounted brain (fluorescent yellow color is DNA dye YO-PRO, which is optional). For electrode localization we use autofluorescence.
To image tissue autofluorescence use 488 nm laser. To image CM-Dil tracks use 561 nm laser. Brain can be imaged with a voxel size of 1.22 µm x 1.22 µm x 8 µm on Z1 with a 5x objective.