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Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow
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Beta-galactosidase staining on bone marrow. The osteoclast pitfall.

H-G Kopp1, A T Hooper, S V Shmelkov

  • 1Department of Genetic Medicine, Howard Hughes Medical Institute, Weill Medical College of Cornell University, New York, New York, USA. hans-georg.kopp@med.uni-tuebingen.de

Histology and Histopathology
|May 25, 2007
PubMed
Summary

Endogenous beta-galactosidase activity in osteoclasts creates a blue background in bone marrow sections. This can be mistaken for transgenic cells, necessitating careful controls in research.

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Area of Science:

  • Biomedical research
  • Histochemistry
  • Cell biology

Background:

  • Beta-galactosidase (lac-Z) is a reporter enzyme used in cell tracking and gene expression studies.
  • Endogenous beta-galactosidase activity can confound results in soft tissues.
  • Background staining in bone marrow sections using X-Gal has not been previously characterized.

Purpose of the Study:

  • To characterize the endogenous beta-galactosidase background staining in mouse bone marrow sections.
  • To determine if osteoclasts exhibit beta-galactosidase activity detectable by X-Gal staining.
  • To assess the potential for confusion between endogenous staining and transgenic reporter expression.

Main Methods:

  • Histochemical staining of mouse bone marrow sections using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl beta-D-Galactoside (X-Gal).

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  • Evaluation of staining patterns from post-partum to adult stages.
  • Testing variations in X-Gal staining procedures (pH, incubation time).

    • Main Results:

    • Osteoclasts in bone marrow sections exhibit specific and robust blue staining with X-Gal.
    • This endogenous staining is present from birth throughout adulthood in mice.
    • Variations in staining protocols did not eliminate the osteoclast background signal.

    Conclusions:

    • Endogenous beta-galactosidase activity in osteoclasts presents a significant background in X-Gal stained bone marrow.
    • This background can be misinterpreted as transgenic reporter gene expression in hematopoietic or stromal cells.
    • Strict controls, including immunohistochemistry or co-staining for specific cell markers, are essential for accurate interpretation of beta-galactosidase activity in bone marrow research.