Array tomography is a new approach to high resolution imaging of protein localization in intact tissue devised by Stephen Smith's laboratory at Stanford. This core will provide project scientists with protocols, reagents, scientific advice, technical assistance, equipment and software needed to carry out Array Tomographic analyses of the localization of synaptic and cellular protein complexes. Array Tomography is quickly becoming the imaging technique of choice for probing synapse density and structure using immuno-fluorescence in mammalian brain sections. Major benefits over traditional immunohistochemistry include greater antibody penetration in ultrathin sections, the possibility of obtaining ultrastructural information from the tissue using electron microscopy after immunofluorescence imaging, and perhaps most importantly for our purposes, the hydrophilic LR White resin, coupled with the ultra-thin tissue sections, allows multiple rounds of antibody staining of the same tissue section. Repeated staining with different antibodies will allow much more detailed study of the co-localization and activation state of signaling molecules and complexes within the soma, nucleus, or synapses of individual neurons. Because relationships between these elements can be characterized within a single cell, non-linear relationships that would be lost upon averaging can be captured. Heterogeneity between cells, something which is likely highly biologically relevant, can be identified and quantified. Only array tomography gives us the technical ability to carry out these kinds of analysis. Pattern of Core usage: tissue preparation, embedding and staining will be performed by individual project scientists with advice and training as needed from experienced core users and/or core staff. Thin sectioning will be performed by a trained technician in the core facility, and automated imaging and image processing will be performed by project scientists working with core staff in the facility. This will maximize flexibility in terms of tissue and antibodies used while maximizing cost effectiveness and data quality by allowing project scientists to share a single microscope and ultramicrotome staffed by technical and scientific personnel dedicated to perfecting the histological, optical and computational aspects of the technique. Administrative structure of the Array Tomography Core A is described in the Administrative Core B.
The projects supported by this Core will begin to illuminate the dynamics of signaling pathways that underlie homeostatic compensation. As defects in homeostatic signaling contribute to neurological syndromes ranging from Autism Spectrum Disorders to Epilepsies, the ability to understand and manipulate homeostatic signaling pathways will be of enormous health relevance.
Showing the most recent 10 out of 15 publications
