ABSTRACT
Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.
Subject(s)
Molecular Imaging/methods , Tissue Preservation/methods , Algorithms , Animals , Female , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Fibers, Myelinated/chemistry , Proteomics , Reducing Agents , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1-3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.
Subject(s)
Antibodies/chemistry , Coloring Agents , Models, Biological , Models, Chemical , Animals , Coloring Agents/chemistry , Coloring Agents/pharmacokinetics , Electrochemical Techniques , Mice , PorosityABSTRACT
The ability of injured axons to regenerate declines with age, yet the mechanisms that regulate axon regeneration in response to age are not known. Here we show that axon regeneration in aging C. elegans motor neurons is inhibited by the conserved insulin/IGF1 receptor DAF-2. DAF-2's function in regeneration is mediated by intrinsic neuronal activity of the forkhead transcription factor DAF-16/FOXO. DAF-16 regulates regeneration independently of lifespan, indicating that neuronal aging is an intrinsic, neuron-specific, and genetically regulated process. In addition, we found that DAF-18/PTEN inhibits regeneration independently of age and FOXO signaling via the TOR pathway. Finally, DLK-1, a conserved regulator of regeneration, is downregulated by insulin/IGF1 signaling, bound by DAF-16 in neurons, and required for both DAF-16- and DAF-18-mediated regeneration. Together, our data establish that insulin signaling specifically inhibits regeneration in aging adult neurons and that this mechanism is independent of PTEN and TOR.
