Light microscopy methods allow studying biological objects at very small depth. Therefore more or less large of biological objects are impossible for imaging as a whole without making damage to its structure. Recently several methods have been proposed for solving this problem: one makes the sample transparent for visible light but still not permeable for macromolecules such as antibodies. The other technique named CLARITY, was developed for immunochemistry studying neuronal circuits and synaptic connections between neurons in the whole brain of adult mice. Here we provided an optimized original CLARITY protocol for immunochemical studying of clarified mouse brain. We have simplified and shortened the protocol in several steps including perfusion, immunostaining and imaging. We have demonstrated immunostaining with antibodies against post-synaptic density protein PSD-95 and striatal specific DARPP32 protein in brain slices. Moreover brain tissue from Thy1-GFP mouse (M-line) was clarified, immunostained and analyzed with further 3D reconstruction of neuronal structures of hippocampus.
This work was supported by the contract with the Russian Ministry of Science 11.G34.31.0056 (Ilya Bezprozvanny) and NIH grants R01NS074376 and R01NS080152 (I.B.).