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In Situ Hybridization to Identify Gut Stem Cells

Alex Gregorieff1,  Hans Clevers1

1Hubrecht Institute, Utrecht, The Netherlands

Publication Name: 
Current Protocols in Stem Cell Biology
Unit Number: 
Unit 2F.1
DOI: 
10.1002/9780470151808.sc02f01s12
Online Posting Date: 
March, 2010
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Abstract

In recent years, considerable effort has been directed towards identifying the repertoire of genes specifically expressed in adult stem cells. In this unit, we describe an in situ hybridization protocol adapted for the analysis of gene expression in the intestinal mucosa. This methodology allows researchers to quickly visualize the expression profile of putative stem cell markers with a high degree of sensitivity and resolution. Curr. Protoc. Stem Cell Biol. 12:2F.1.1-2F.1.11. © 2010 by John Wiley & Sons, Inc.

Keywords: (ISH) in situ hybridization; digoxigenin RNA probes; formalin-fixed paraffin-embedded; intestinal sections

     
 
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Table of Contents

  • Introduction
  • Basic Protocol: In Situ Hybridization to Detect Stem Cell Genes
  • Support Protocol: Generation of Digoxigenin RNA Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol: In Situ Hybridization to Detect Stem Cell Genes

 Materials
  • Rat or mouse intestine, freshly dissected
  • 10% (v/v) neutral buffered formalin (fixative)
  • Phosphate-buffered saline (PBS; see recipe)
  • 25%, 50%, 75%, 90%, and 100% (v/v) ethanol
  • Xylenes
  • Paraffin wax
  • Absolute ethanol, 96% ethanol
  • DEPC-treated H2O
  • HCl
  • Proteinase K
  • Glycine
  • Paraformaldehyde (see recipe)
  • Acetic anhydride solution (see recipe)
  • 20× SSC, pH 4.5 (see recipe)
  • 20× SSC, pH 7.5 (see recipe)
  • Formamide
  • Hybridization solution (see recipe)
  • Digoxigenin-labeled probe (Support Protocol)
  • Tris/NaCl buffer (see recipe)
  • Blocking solution (see recipe)
  • Anti-digoxigenin AP-conjugated antibody (Roche)
  • NTM buffer (see recipe)
  • NBT/BCIP (Sigma) working solution (see recipe)
  • Permanent mounting medium
  • Microtome
  • Tweezers
  • 10-ml syringe and 25-G needle
  • Small cardboard rings and pins
  • Histological slides (Superfrost Plus slides)
  • Temperature-regulated oven
  • Glass jars (e.g., Coplin jars with lids)
  • Covered slide box
  • Coverslips
  • Light microscope

Support Protocol: Generation of Digoxigenin RNA Probes

 Materials
  • Plasmid for gene of interest (e.g., see Table 2F.1.3)
  • Restriction endonuclease and buffer (Bloch and Grossman, 1995)
  • Agarose
  • 3 M sodium acetate, pH 5.2
  • Phenol/choloroform
  • Absolute ethanol
  • 70% (v/v) ethanol
  • 10 × transcription buffer (Roche)
  • Dithiothreitol (DTT)
  • 10 × Dig RNA labeling mix (Roche)
  • RNase inhibitor (Fermentas)
  • T7 or T3 or SP6 RNA polymerases (Roche)
  • DEPC-treated H2O
  • Rnase-free DNaseI (Fermentas), optional
  • 4 M LiCl
  • Formamide
  • 1.5-ml microcentrifuge tubes
  • RNA purification columns (RNeasy Mini Kit, Qiagen)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, 2000) and digestion of DNA with restriction enzymes (Bloch and Grossman, 1995)
Looking for materials?  Suppliers Guide
     
 
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Literature Cited

Literature Cited
    Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. 2007. Identification of stem cells in small intestine and colon by marker gene lgr5. Nature 449:1003-1007.
    Bloch, K.D. and Grossmann, B. 1995. Digestion of DNA with restriction endonucleases. Curr. Protoc. Mol. Biol. 31:3.1.1-3.1.21.
    Cheng, H. and Leblond, C.P. 1974a. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am. J. Anat. 141:461-479.
    Cheng, H. and Leblond, C.P. 1974b. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141:537-561.
    Potten, C.S. 1977. Extreme sensitivity of some intestinal crypt cells to x and gamma irradiation. Nature 269:518-521.
    Potten, C.S., Kovacs, L., and Hamilton, E. 1974. Continuous labelling studies on mouse skin and intestine. Cell Tissue Kinet. 7:271-283.
    Potten, C.S., Owen, G., and Booth, D. 2002. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J. Cell Sci. 115:2381-2388.
    Sangiorgi, E. and Capecchi, M.R. 2008. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40:915-920.
    van der Flier, L.G., van Gijn, M.E., Hatzis, P., Kujala, P., Haegebarth, A., Stange, D.E., Begthel, H., van den Born, M., Guryev, V., Oving, I., van Es, J.H., Barker, N., Peters, P.J., van der Wetering, M, and Clevers, H. 2009. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136:903-912.
    Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.
     
 
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