Hubei Provincial Clinical Research Center for Diabetes and Metabolic Disorder

4 ARTICLES PUBLISHED IN JoVE

Developmental Biology

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Nan Zhang^1,2, Liakot A Khan¹, Edward Membreno¹, Gholamali Jafari¹, Siyang Yan¹, Hongjie Zhang^1,3, Verena Gobel¹

¹Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital, Harvard Medical School, ²College of Life Sciences, Jilin University, ³Faculty of Health Sciences, University of Macau

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Developmental Biology

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

Nan Zhang^1,2, Edward Membreno¹, Susan Raj¹, Hongjie Zhang^1,3, Liakot A Khan¹, Verena Gobel¹

¹Mucosal Immunology and Biology Research Center, Developmental Biology and Genetics Core, Massachusetts General Hospital for Children, Harvard Medical School, ²College of Life Sciences, Jilin University, ³Faculty of Health Sciences, University of Macau

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

Environment

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Shan Gao^*1, Weiyang Chen^*2, Nan Zhang¹, Chi Xu³, Haiming Jing^1,4, Wenjing Zhang^1,4, Gaochao Han^1,4, Matthew Flavel⁵, Markandeya Jois⁵, Yingxin Zeng¹, Jing-Dong J. Han³, Bo Xian³, Guojun Li^1,4

¹Beijing Key Laboratory of Diagnostic and Traceability Technologies for Food Poisoning, Beijing Center for Disease Prevention and Control/Beijing Center of Preventive Medicine Research, China, ²College of Computer Science and Technology, Qilu University of Technology(Shandong Academy of Sciences), China, ³Key Laboratory of Computational Biology, CAS Center for Excellence in Molecular Cell Science, Collaborative Innovation Center for Genetics and Developmental Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China, ⁴Beijing Key Laboratory of Environmental Toxicology, School of Public Health, Capital Medical University, China, ⁵School of Life Sciences, La Trobe University, Australia

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Neuroscience

Free-floating Immunostaining of Mouse Brains

Longlong Tu¹, Nan Zhang^1,2,3, Kristine M Conde¹, Jonathan C Bean¹, Chunmei Wang¹, Yong Xu^1,4

¹USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, ²Department of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, ³Hubei Provincial Clinical Research Center for Diabetes and Metabolic Disorder, ⁴Department of Molecular and Cellular Biology, Baylor College of Medicine

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.