ß-Galactosidase staining on bone marrow. The osteoclast pitfall
H.-G. Kopp1,2,*, A.T. Hooper1,*, S.V. Shmelkov1 and S. Rafii1
1Department of Genetic Medicine, Howard Hughes Medical Institute, Weill Medical College of Cornell University, New York, New York, USA, 2Eberhard-Karls University Tubingen, Department of Hematology-Oncology, Germany
Offprint requests to: Hans-Georg Kopp, MD, Eberhard-Karls University Tubingen, Department of Hematology-Oncology, Otfried-Muller Str. 10, 72076 Tubingen, Germany. e-mail: hans-georg.kopp@med.uni-tuebingen.de.
*H.-G.K. and A.T.H. have contributed equally to this work
Summary. 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 ß-galactosidase activity. Previously, endogenous ß-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 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 ß-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 ß-galactosidase positive bone marrow cells. Verifiable bacterial ß-galactosidase positive bone marrow cells should be further identified using immunohistological or other approaches. Specifically, ß-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. Histol Histopathol 22, 971-976 (2007)
Key words: ß-galactosidase, X-Gal staining, Bone marrow, Osteoclasts
DOI: 10.14670/HH-22.971