Cite as: Cold Spring Harb. Protoc.; 2007; doi:10.1101/pdb.prot4822

This Protocol
Right arrow Full Text
Right arrow Update/discuss this protocolDiscussion icon
Right arrow Alert me when this protocol is cited
Right arrow Alert me when comments are published
Right arrow Alert me if a correction is posted
Services
Right arrow Similar protocols in this database
Right arrow Alert me to new releases of protocols
Right arrow Save to Personal Folders
Right arrow Download to citation manager
Right arrow Printer-friendly versionPrinter-friendly version
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lufkin, T.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Lufkin, T.
Related Collections
Right arrow CSHL Molecular Embryology of the Mouse Course
Right arrow Cell Biology, general
Right arrow Visualization
Right arrow Imaging Development
Right arrow Laboratory Organisms, general
Right arrow Mouse
Right arrow Molecular Biology, general
Right arrow Analysis of Gene Expression
Right arrow Probes
Right arrow Probes, general
Right arrow Non-isotopically Labeled Probes
Right arrow RNA
Right arrow RNA, general
Right arrow mRNA
Right arrow Developmental Biology
Right arrow In Situ Hybridization
Right arrow Visualization of Gene Expression
Right arrowRelated Article
Right arrowRelated Protocols
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?
BSN globe

protocolProtocol

In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Preparation of Embryos and Probes

Thomas Lufkin

This protocol was adapted from "Techniques for Visualizing Gene Products, Cells, Tissues, and Organ Systems," Chapter 16, in Manipulating the Mouse Embryo, 3rd edition, by Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2003.


INTRODUCTION

This is the first of two protocols describing how to perform in situ hybridization on whole mouse embryos. Here we describe how to fix and permeabilize embryos with detergent and how to synthesize single-stranded RNA probes (riboprobes) in vitro using UTP labeled with the plant steroid digoxigenin (DIG-11-UTP). Antisense digoxigenin-labeled riboprobes are synthesized as run-off transcripts from linearized plasmid templates using bacteriophage RNA polymerases (T3, T7, SP6) under standard conditions. The probe is precipitated with ethanol in the presence of NaCl, and after resuspension in DEPC-treated H2O is stable at –20°C for many months. Probes ranging from 250 to 1500 bp have been used successfully. The optimal probe concentration is in the 100 ng to 1 µg/mL range and should be determined empirically.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?

Related Article

General Procedures for Avoiding Contamination with RNase
Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
CSH Protocols 2007: 43. [Extract] [Full Text]

Related Protocols

Agarose Gel Electrophoresis
Joseph Sambrook and David W. Russell
CSH Protocols 2006: 4020. [Extract] [Full Text]

Handling Mouse Blastocysts for Fixation
Andras Nagy, Marina Gertsenstein, Kristina Vintersten, and Richard Behringer
CSH Protocols 2007: 4814. [Abstract] [Full Text]

In Situ Hybridization of Whole-Mount Mouse Embryos with RNA Probes: Hybridization, Washes, and Histochemistry
Thomas Lufkin
CSH Protocols 2007: 4823. [Abstract] [Full Text]



This article has been cited by other articles:


Home page
 CSH ProtocolsHome page
P. K. Dearden, E. J. Duncan, and M. J. Wilson
Whole-Mount In Situ Hybridization of Honeybee (Apis mellifera) Tissues
CSH Protocols, June 1, 2009; 2009(6): pdb.prot5225 - pdb.prot5225.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
P. K. Dearden, E. J. Duncan, and M. J. Wilson
In Situ Hybridization of Sectioned Honeybee (Apis mellifera) Tissues
CSH Protocols, June 1, 2009; 2009(6): pdb.prot5226 - pdb.prot5226.
[Abstract] [Full Text]

Home page
 CSH ProtocolsHome page
B. Rumballe, K. Georgas, and M. H. Little
High-Throughput Paraffin Section In Situ Hybridization and Dual Immunohistochemistry on Mouse Tissues
CSH Protocols, July 1, 2008; 2008(8): pdb.prot5030 - pdb.prot5030.
[Abstract] [Full Text]