Whole‐Mount Histochemical Detection of β‐Galactosidase Activity

Fred A. Pereira1

1 Baylor College of Medicine, Houston, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.14
DOI:  10.1002/0471142727.mb1414s50
Online Posting Date:  May, 2001
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Abstract

This unit describes fixation and staining for b‐galactosidase activity; it has been successfully used on vertebrate embryos and tissue explants. To help achieve consistent results with larger samples, an alternate protocol details preparation of thick sections. Support protocols are provided for clearing of tissues for better resolution and paraffin‐embedding the whole embryo.

     
 
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Table of Contents

  • Basic Protocol 1: Whole‐Mount Staining and Histochemical Detection of β‐Galactosidase Activity
  • Alternate Protocol 1: Preparing Thick Sections of Large Tissues for β‐Galactosidase Staining
  • Support Protocol 1: Storage and Tissue Clearing
  • Support Protocol 2: Paraffin Embedding, Sectioning, and Counter‐Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Whole‐Mount Staining and Histochemical Detection of β‐Galactosidase Activity

  Materials
  • Mouse or chicken whole embryos, tissues, or explants
  • Phosphate‐buffered saline (PBS; appendix 22), ice‐cold
  • recipeβ‐galactosidase fixative (see recipe)
  • recipeβ‐galactosidase wash buffer (see recipe)
  • recipeβ‐galactosidase staining solution (see recipe)
  • 5‐ml or 10‐ml polystyrene or polypropylene capped tubes or 1.5‐ to 2‐ml microcentrifuge tubes
  • Platform rocker
  • Sealable container for staining
  • Depression slides with coverslips or chamber slides
  • Dissecting microscope with fiber‐optic lighting
  • Photomicrography equipmentNOTE: All steps are performed in 5‐ml, 10‐ml polystyrene or polypropylene capped tubes or in 1.5‐ to 2‐ml microcentrifuge tubes in solutions (20:1 v/v reagent‐to‐tissue ratio) with gentle end‐to‐end rocking unless stated otherwise.

Alternate Protocol 1: Preparing Thick Sections of Large Tissues for β‐Galactosidase Staining

  • recipe4% agarose (see recipe)
  • Cyanoacrylate glue (e.g., Superglue)
  • 100 mM sodium phosphate buffer, pH 8 ( appendix 22)
  • Vibratome (e.g., model VT1000S; Leica)

Support Protocol 1: Storage and Tissue Clearing

  Materials
  • Stained samples (see protocol 1Basic and protocol 2)
  • 3.7% formaldehyde: dilute 37% formaldehyde 1:10 with PBS
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 50%, 70%, 80%, 95%, and 100% methanol
  • 2:1 (v/v) benzyl benzoate/benzyl alcohol

Support Protocol 2: Paraffin Embedding, Sectioning, and Counter‐Staining

  Materials
  • Whole‐mount stained embryos (see protocol 1)
  • recipe3% agarose (see recipe)
  • Histoclear (National Diagnostics)
  • 70%, 95%, and 100% ethanol
  • 0.1% (w/v) nuclear fast red in 5% (w/v) aqueous aluminum sulfate: (Kernechtrot; Poly Scientific)
  • Permount or aqueous mounting medium
  • Microtome
  • Subbed microscope slides (e.g., Superfrost Plus, Fisher)
  • 42°C and 65°C ovens
  • Coplin jars or staining dishes
  • Photomicrography equipment
  • Additional reagents and equipment for paraffin embedding (unit 14.1)
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Figures

Videos

Literature Cited

Literature Cited
   Beddington, R.S.P. and Lawson, K.A. 1990. Clonal analysis of cell lineages. In Postimplantation Mammalian Embryos: A Practical Approach (A.J. Copp and D.L. Cockroft, eds.) pp. 267‐292. IRL Press, Oxford.
   Bonnerot, C., Rocancourt, D., Briand, P., Grimber, G., and Nicolas., J.F. 1987. A β‐galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc. Natl. Acad. Sci. U.S.A. 84:6795‐6799.
   Collignon, J., Varlet, I., and Robertson, E.J. 1996. Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381:155‐158.
   Hogan, B., Beddington, R., Constantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo, 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, New York.
   Lis, J.T., Simon, J.A., and Sutton, C.A. 1983. New heat shock puffs and beta‐galactosidase activity resulting from transformation of Drosophila with an hsp70‐lacZ hybrid gene. Cell 35:403‐410.
   MacGregor, G.R., Mogg, A.E., Burke, J.F., and Caskey, C.T. 1987. Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene. Somatic Cell Mol. Genet. 13:253‐265.
   Martineau, J., Nordqvist, K., Tilmann, C., Lovell‐Badge, R., and Capel, B. 1997. Male‐specific cell migration into the developing gonad. Curr. Biol. 7:958‐968.
   Muller‐Hill, B. and Kania, J. 1974. Lac repressor can be fused to beta‐galactosidase. Nature 249:561‐563.
   Naya, F.J., Huang, H.P., Qiu, Y., Mutoh, H., DeMayo, F.J., Leiter, A.B., and Tsai, M.J. 1997. Diabetes, defective pancreatic morphogenesis, and abnormal enteroendocrine differentiation in BETA2/neuroD‐deficient mice. Genes Dev. 11:2323‐2334.
   Partanen, J., Puri, M.C., Schwartz, L., Fischer, K.D., Bernstein, A., and Rossant, J. 1996. Cell autonomous functions of the receptor tyrosine kinase TIE in a late phase of angiogenic capillary growth and endothelial cell survival during murine development. Development 122:3013‐3021.
   Pereira, F.A., Qiu, Y., Zhou, G., Tsai, M.J., and Tsai, S.Y. 1999. The orphan nuclear receptor COUP‐TFII is required for angiogenesis and heart development. Genes Dev. 13:1037‐1049.
   Sanes, J.R., Rubenstein, J.L., and Nicolas, J.F. 1986. Use of a recombinant retrovirus to study post‐implantation cell lineage in mouse embryos. EMBO J. 5:3133‐3142.
   Silhavy, T.J. and Beckwith, J.R. 1985. Uses of lac fusions for the study of biological problems. Microbiol. Rev. 49:398‐418.
   Sullivan, R. and Lo, C.W. 1997. Histochemical and fluorochrome‐based detection of β‐galactosidase. In Methods in Molecular Biology, Vol. 63: Recombinant Protein Protocols: Detection and Isolation (R. Tuan, ed.), Humana Press Inc., Totowa, New Jersey.
   Wang, H.U., Chen, Z.F., and Anderson, D.J. 1998. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin‐B2 and its receptor Eph‐B4. Cell 93:741‐753.
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