The online version of this article (doi:10.1186/1750-1326-9-19) contains supplementary material, which is available to authorized users.
Ekaterina Poguzhelskaya, Dmitry Artamonov contributed equally to this work.
The authors declare that they have no competing interests.
EP and DA performed the experiments. EP, DA, AB analyzed data. EP, DA, OV and IB designed research. EP, DA, AB, OV, IB wrote the paper and prepare figures for publication. All authors read and approved the final manuscript.
Imaging methods are used widely to understand structure of brain and other biological objects. However, sample penetration by light microscopy is limited due to light scattering by the tissue. A number of methods have been recently developed to solve this problem. In one approach (SeeDB) simple procedure for clarifying brain samples for imaging was described. However, this method is not compatible with immunostaining approach as SeeDB-prepared tissue is not permeable to the antibodies. Another technique for clearing brain tissue (CLARITY) was optimized for immunochemistry, but this method technically much more demanding than SeeDB.
Here we report optimized protocol for imaging of brain samples (CLARITY2). We have simplified and shortened the original protocol. Following hydrogel fixation, we cut brain tissue to 1–1.5 mm thick coronal slices. This additional step enabled us to accelerate and simplify clearing, staining and imaging steps when compared to the original protocol. We validated the modified protocol in imaging experiments with brains from line M Thy1-GFP mouse and in immunostaining experiments with antibodies against postsynaptic protein PSD-95 and striatal-specific protein DARPP32.
The original CLARITY protocol was optimized and simplified. Application of the modified CLARITY2 protocol could be useful for a broad range of scientists working in neurobiology and developmental biology.
Additional file 1: 3D reconstruction of hippocampal neurons form line M Thy1-GFP mice based on two-photon imaging data (20 x objective) .(MP4 2 MB)13024_2014_531_MOESM1_ESM.mp4
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- Simplified method to perform CLARITY imaging
- BioMed Central
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