ß-Galactosidase staining on bone marrow. The osteoclast pitfall

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.