In Situ Hybridization to Identify Gut Stem Cells

Alex Gregorieff1, Hans Clevers1

1 Hubrecht Institute, Utrecht
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2F.1
DOI:  10.1002/9780470151808.sc02f01s34
Online Posting Date:  August, 2015
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In recent years, considerable effort has been directed toward 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. © 2015 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 1: In Situ Hybridization to Detect Stem Cell Genes
  • Support Protocol 1: Generation of Digoxigenin RNA Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
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Basic Protocol 1: In Situ Hybridization to Detect Stem Cell Genes

  • 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 H 2O
  • 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 ( protocol 2Support 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 1: Generation of Digoxigenin RNA Probes

  • Plasmid for gene of interest (e.g., see Table 2.1.3)
  • Restriction endonuclease and buffer (Bloch and Grossmann, )
  • 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 H 2O
  • 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, ) and digestion of DNA with restriction enzymes (Bloch and Grossmann, )
Table 2.0.3   MaterialsUseful Epithelial, Cell‐Type Specific Markers for In Situ Hybridization in the Small Intestine

Cell type Gene
Enterocytes Fabp1, Fabp2
Goblet cells Gob5, Tff3
Enteroendocrine cells Chromogranin B
Paneth cells Cryptdins
Entire epithelium Villin, Tcf4
Proliferative crypt epithelial cells c‐Myc, c‐Myb
Early progenitors/stem cells Axin2, Lgr5, Olfm4

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Literature Cited

Literature Cited
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   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.
   Itzkovitz, S. , Lyubimova, A. , Blat, I.C. , Maynard, M. , van Es, J. , Lees, J. , Jacks, T. , Clevers, H. , and van Oudenaarden, A. 2011. Single‐molecule transcript counting of stem‐cell markers in the mouse intestine. Nat. Cell Biol. 14:106‐114.
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   Sangiorgi, E. and Capecchi, M.R. 2008. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40:915‐920.
   Takeda, N. , Jain, R. , LeBoeuf, M.R. , Wang, Q. , Lu, M.M. , and Epstein, J.A. 2011. Interconversion between intestinal stem cell populations in distinct niches. Science 334:1420‐1424.
   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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