Abstract
Tissue clearing methods promise to provide exquisite three-dimensional imaging information; however, there is a need for simplified methods for lower resource settings and for non-fluorescence based phenotyping to enable light microscopic imaging modalities. Here we describe the simplified CLARITY method (SCM) for tissue clearing that preserves epitopes of interest. We imaged the resulting tissues using light sheet microscopy to generate rapid 3D reconstructions of entire tissues and organs. In addition, to enable clearing and 3D tissue imaging with light microscopy methods, we developed a colorimetric, non-fluorescent method for specifically labeling cleared tissues based on horseradish peroxidase conversion of diaminobenzidine to a colored insoluble product. The methods we describe here are portable and can be accomplished at low cost, and can allow light microscopic imaging of cleared tissues, thus enabling tissue clearing and imaging in a wide variety of settings.
Highlights
We describe a more portable implementation of tissue clearing, based on passive CLARITY technique (PACT), termed simplified CLARITY method (SCM)
We show that we can utilize colorimetric methods for specific labeling and imaging of tissues based on the diaminobenzidine reaction and the resulting tissues can be imaged with visible light microscopy techniques
We utilized light sheet microscopy (LSM) to image fluorescent labels within intact cleared tissues, to enable the deepest tissue imaging possible at a fast rate, as we have previously shown that LSM offers the greatest depth of view and most rapid image acquisition as compared to commercially available confocal microscopy systems[10]
Summary
We describe a more portable implementation of tissue clearing, based on PACT, termed SCM (simplified CLARITY method). We show that polymerization and clearing can be performed with minimal equipment. We show that we can utilize colorimetric methods for specific labeling and imaging of tissues based on the diaminobenzidine reaction and the resulting tissues can be imaged with visible light microscopy techniques. These methods hold promise to enable tissue processing, clearing, and imaging at reduced cost and to enable additional, complementary microscopic methods to be utilized to image the resulting tissues
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