Publication: ß-Galactosidase staining on bone marrow. The osteoclast pitfall
Authors
Kopp, H.G. ; Hooper, A.T. ; Shmelkov, S.V. ; Rafii, S.
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Publisher
Murcia : F. Hernández
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DOI
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info:eu-repo/semantics/article
Description
Abstract
The enzyme ß-galactosidase, encoded by the
bacterial gene lac-Z, is commonly used as a
histochemical reporter to track transplanted cells in vivo
or to analyze temporospatial gene expression patterns by
coupling expression of specific target genes to ßgalactosidase
activity. Previously, endogenous ßgalactosidase
activity has been recognized as a
confounding factor in the study of different soft tissues,
but there is no description of the typical background on
bone marrow sections when using the chromogenic
substrate 5-Bromo-4-chloro-3-indolyl ß-D-Galactoside
(X-Gal). In this report, we show that osteoclasts in bone
marrow sections specifically and robustly stain blue with
X-Gal. This leads to a typical background when bone
marrow is examined that is present from the first day
post partum throughout the adult life of experimental
mice and can be confused with transgenic, bacterial ßgalactosidase
expressing hematopoietic or stromal cells.
Experimental variations in the X-Gal staining procedure,
such as pH and time of exposure to substrate, were not
sufficient to avoid this background. Therefore, these data
demonstrate the need for strenuous controls when
evaluating ß-galactosidase positive bone marrow cells.
Verifiable bacterial ß-galactosidase positive bone
marrow cells should be further identified using
immunohistological or other approaches. Specifically, ßgalactosidase
positive hematopoietic or stromal cells
should be proven specifically not to be osteoclasts by costaining
or staining adjacent sections for specific
markers of hematopoietic and stromal cells.
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