Reduction of Photo Bleaching and Long Term Archiving of Chemically Cleared GFP-Expressing Mouse Brains

Tissue clearing allows microscopy of large specimens as whole mouse brains or embryos. However, lipophilic tissue clearing agents as dibenzyl ether limit storage time of GFP-expressing samples to several days and do not prevent them from photobleaching during microscopy. To preserve GFP fluorescence, we developed a transparent solid resin formulation, which maintains the specimens' transparency and provides a constant signal to noise ratio even after hours of continuous laser irradiation. If required, high-power illumination or long exposure times can be applied with virtually no loss in signal quality and samples can be archived for years.

[1]  N. Hardie A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy , 2004 .

[2]  R. Weiler,et al.  Chemical Clearing and Dehydration of GFP Expressing Mouse Brains , 2012, PloS one.

[3]  A. Schierloh,et al.  Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain , 2007, Nature Methods.

[4]  W. Lockwood,et al.  A reliable and easily sectioned epoxy embedding medium , 1964, The Anatomical record.

[5]  S. Hell,et al.  Nanoscale resolution in GFP-based microscopy , 2006, Nature Methods.

[6]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[7]  J. Swedlow,et al.  Evaluating performance in three-dimensional fluorescence microscopy , 2007, Journal of microscopy.

[8]  John H. Luft,et al.  IMPROVEMENTS IN EPOXY RESIN EMBEDDING METHODS , 1961, The Journal of biophysical and biochemical cytology.

[9]  L. Luciano,et al.  Re-evaluation of Epoxy Resin Sections for Light and Electron Microscopic Immunostaining , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[10]  A. Spurr A low-viscosity epoxy resin embedding medium for electron microscopy. , 1969, Journal of ultrastructure research.

[11]  S. Qadri,et al.  Oxyradical-induced GFP damage and loss of fluorescence. , 2008, International journal of biological macromolecules.

[12]  Valery V. Tuchin,et al.  Optical clearing of tissues and blood using the immersion method , 2005 .

[13]  S. Brorson The combination of high-accelerator epoxy resin and antigen retrieval to obtain more intense immunolabeling on epoxy sections than on LR-white sections for large proteins. , 1998, Micron.

[14]  E. Rubel,et al.  A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy , 2004, Brain Research.

[15]  J A Dent,et al.  A whole-mount immunocytochemical analysis of the expression of the intermediate filament protein vimentin in Xenopus. , 1989, Development.