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Ultrafast tissue staining with chemical tags.

ABSTRACT: Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.

SUBMITTER: Kohl J

PROVIDER: S-EPMC4246963 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Ultrafast tissue staining with chemical tags.

Kohl Johannes J Ng Julian J Cachero Sebastian S Ciabatti Ernesto E Dolan Michael-John MJ Sutcliffe Ben B Tozer Adam A Ruehle Sabine S Krueger Daniel D Frechter Shahar S Branco Tiago T Tripodi Marco M Jefferis Gregory S X E GS

Proceedings of the National Academy of Sciences of the United States of America 20140825 36

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background lab ...[more]

PMID: 25157152

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Project description:Diffusible iodine-based contrast-enhanced computed tomography (diceCT) techniques allow visualization of soft tissues of fluid-preserved specimens in three dimensions without dissection or histology. Two popular diceCT stains, iodine-potassium iodide (I2KI) dissolved in water and elemental iodine (I2) dissolved in 100% ethanol (EtOH), yield striking results. Despite the widespread use of these stains in clinical and biological fields, the molecular mechanisms that result in color change and radiopacity attributed to iodine staining are poorly understood. Requests to apply these stains to anatomical specimens preserved in natural history museums are increasing, yet curators have little information about the potential for degradation of treated specimens. To assess the molecular effects of iodine staining on typical museum specimens, we compared the two popular stains and two relatively unexplored stains (I2KI in 70% EtOH, I2 in 70% EtOH). House sparrows (Passer domesticus) were collected and preserved under uniform conditions following standard museum protocols, and each was then subjected to one of the stains. Results show that the three ethanol-based stains worked equally well (producing fully stained, life-like, publication quality scans) but in different timeframes (five, six, or eight weeks). The specimen in I2KI in water became degraded in physical condition, including developing flexible, demineralized bones. The ethanol-based methods also resulted in some demineralization but less than the water-based stain. The pH of the water-based stain was notably acidic compared to the water used as solvent in the stain. Our molecular analyses indicate that whereas none of the stains resulted in unacceptable levels of protein degradation, the bones of a specimen stained with I2KI in water demineralized throughout the staining process. We conclude that staining with I2KI or elemental I2 in 70% EtOH can yield high-quality soft-tissue visualization in a timeframe that is similar to that of better-known iodine-based stains, with lower risk of negative impacts on specimen condition.

Dataset Information

Ultrafast tissue staining with chemical tags.

Publications

Ultrafast tissue staining with chemical tags.

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