The E.coli gene Lac-Z encodes for β-galactosidase and is often used to track transgenic cells after transplantation in vivo or to analyze temporospatial gene expression patterns by coupling expression of specific target genes to β-galactosidase activity. Several groups have reported endogenous β-galactosidase activity as a confounding factor in the study of different soft tissues. However, the typical background to be found on bone marrow sections when using the chromogenic substrate 5-Bromo-4-chloro-3-indolyl β-D-Galactoside (X-Gal) has not been described before. We demonstrate here that X-Gal stained wild type murine bones display a typical epiphyseal β-gal positive stain line that can already be seen on whole mount preparations. With light microscopy, the β-gal positive cells are located along the endosteum adjacent to trabecular bone and in particularly high concentrations in the epiphyseal resorption zone. In addition, the β-gal positive cells bear the typical features of osteoclasts in that they are multinucleated and display the typical foamy cytoplasm of osteoclasts. When an antibody against a well-known and previously published marker of the vasculature, pan-endothelial cell antigen (clone MECA32), was used in wild type bone, the X-Gal positive osteoclasts could be found in their typical location in epiphyseal resorption pits and closely located to the bone marrow vasculature. This provides further evidence that the X-Gal positive cells are indeed osteoclasts. Finally, double staining for tartrate resistant acid phosphatase (TRAP) and with X-Gal in wild type murine bones resulted in a complete match of the staining pattern and provided formal proof of the hypothesis that osteoclasts have strong β-galactosidase activity. In order to establish common knowledge of this potentially misleading artifact, we systematically examined the bones of different wild type mouse strains at various stages of development starting at the neonatal period. The same pattern of background staining was observed in C57bl/6, FvB/NJ, 129P3/J, and CD1 strains of mice. In addition, we examined the effect of pH and time of exposure to substrate and observed no changes in endogenous mammalian b-gal activity.

In conclusion, 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 easily be confused with transgenic, bacterial β-galactosidase expressing hematopoietic or stromal cells. We conclude from these data that there is a field-wide need for strenuous controls and further classification when evaluating β-galactosidase positive bone marrow cells. Verifiable bacterial β-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Alternatively, β-galactosidase positive cells should be proven not to be osteoclasts by co-staining or by staining of adjacent sections.

Disclosure: No relevant conflicts of interest to declare.

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