College of William and Mary View Institution's Website 6 articles published in JoVE Developmental Biology Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis Eileen F. Ablondi1, Sudip Paudel2, Morgan Sehdev3, John P. Marken4, Andrew D. Halleran4, Atiqur Rahman5, Peter Kemper5, Margaret S. Saha2 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Harvard University, 2Department of Biology, College of William and Mary, 3Harvard Medical School, Harvard University, 4Department of Bioengineering, California Institute of Technology, 5Department of Computer Science, College of William and Mary We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level. Biochemistry Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins Rui Yin, Catherine Harvey, Diane C. Shakes, Oliver Kerscher 1Integrated Science Center, Biology Department, College of William & Mary SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types. Biology Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ Ashley Fidler*1, Lauren Boulay*1, Matthew Wawersik1 1Department of Biology, College of William & Mary Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae. Biology Dissection and Downstream Analysis of Zebra Finch Embryos at Early Stages of Development Jessica R. Murray1, Monika E. Stanciauskas1, Tejas S. Aralere1, Margaret S. Saha1 1Department of Biology, College of William and Mary The zebra finch (Taeniopygiaguttata) is a valuable model organism; however, early stages of zebra finch development have not been extensively studied. The protocol describes how to dissect early embryos for developmental and molecular applications. Biology Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications Eva Paige Szymanski1, Oliver Kerscher1 1Integrated Science Center, Biology Department, The College of William & Mary The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications. Neuroscience Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue Molly J. McDonough*1, Chelsea E. Allen*1, Ng-Kwet-Leok A. Ng-Sui-Hing1, Brian A. Rabe1, Brittany B. Lewis1, Margaret S. Saha1 1Department of Biology, College of William and Mary Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.
