Biology Reference
In-Depth Information
CHAPTER 9
In situ
Hybridization of Embryos with
Antisense RNA Probes
Gina Broitman-Maduro and Morris F. Maduro
Department of Biology, University of California at Riverside, Riverside, California, USA
Abstract
I. Introduction
II. Rationale
III. Methods
A. Probe Design and Synthesis
B. PCR to Generate Probe Synthesis Template
C. DIG-Labeled Probe Synthesis
IV. Animal Preparation and Fixation
A. Synchronization to Produce Gravid Hermaphrodites
B. Fixation of Gravid Hermaphrodites and Embryos
C. Freeze-Cracking and Fixation
D. Hydration Series
V. Hybridization and Signal Development
A. Prehybridization and Probe Hybridization
B. Rinsing and Antibody Incubation
C. Rinsing and Signal Development
VI. Materials
VII. Notes
A. Suggested Positive Controls
B. Hypaque meglumine
C. Nontoxic Fixative
D. Purchase of Ready-Made Reagents
E. Labeling of Nuclei
F. Staining Small Quantities of Embryos or Other Stages
G. Staining of Other Species
VIII. Troubleshooting Guide
IX. Imaging of Stained Embryos
X. Summary
Acknowledgments
References
 
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