Morgridge Institute for Research View Institution's Website 4 articles published in JoVE Biochemistry Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows Bryan S. Sibert*1,2,3, Joseph Y. Kim*1,4, Jie E. Yang1,2,3, Elizabeth R. Wright1,2,3,5 1Department of Biochemistry, University of Wisconsin, Madison, 2Cryo-Electron Microscopy Research Center, Department of Biochemistry, University of Wisconsin, Madison, 3Midwest Center for Cryo-Electron Tomography, Department of Biochemistry, University of Wisconsin, Madison, 4Department of Chemistry, University of Wisconsin, Madison, 5Morgridge Institute for Research The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding. Biology Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos Anjalie Schlaeppi1, Alyssa Graves1, Michael Weber1, Jan Huisken1 1Morgridge Institute for Research, Madison, WI We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided. Bioengineering Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools Yuming Liu1, Kevin W. Eliceiri1,2,3 1Laboratory for Optical and Computational Instrumentation, Center for Quantitative Cell Imaging, University of Wisconsin-Madison, 2Departments of Medical Physics and Biomedical Engineering, University of Wisconsin-Madison, 3Morgridge Institute for Research, Madison, Wisconsin, 4test, test Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures. Biology Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA Srikumar Sengupta1, Jennifer M. Bolin1, Victor Ruotti1, Bao Kim Nguyen1, James A. Thomson1,2,3, Angela L. Elwell1, Ron Stewart1 1Regenerative Biology, Morgridge Institute for Research, 2Department of Cell & Regenerative Biology, University of Wisconsin, 3Department of Molecular, Cellular, & Regenerative Biology, University of California Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.
