β-galactosidase staining on bone marrow.: The osteoclast pitfall

The enzyme beta-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 beta-galactosidase activity. Previously, endogenous beta-galactosidase 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 beta-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 beta-galactosidase 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 beta-galactosidase positive bone marrow cells. Verifiable bacterial beta-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Specifically, beta-galactosidase positive hematopoietic or stromal cells should be proven specifically not to be osteoclasts by co-staining or staining adjacent sections for specific markers of hematopoietic and stromal cells.