Beta-galactosidase detection as an indicator of endogenous PTHrP in cartilage.
Genetically altered mice are an important tool for biomedical research. Several transgenic mice have been created in which activation of the transgene results in production of beta-galactosidase that can be detected by histological means. While preservation and subsequent visualization of enzyme activity in soft tissues can be complicated, it is particularly difficult in bone specimens, especially those that have been decalcified. For these studies, we examined the bones of parathyroid hormone-related peptide (PTHrP) knock-in mice in which expression of PTHrP resulted in beta-galactosidase production. During the past decade, several studies have demonstrated the importance of PTHrP in bone. Thus, it is important to preserve and detect beta-galactosidase enzymatic activity in bone for these studies. We demonstrate here that beta-galactosidase was visualized better in slides with bone sections taken from PTHrP knock-in mice when bones were frozen and sectioned compared to bones that were embedded in plastic and sectioned using a microtome. Importantly, we were able to visualize beta-galactosidase in plastic embedded bones when specimens were fixed, stained (X-gal), embedded in plastic, and then sectioned rather than being fixed, embedded in plastic, sectioned, then stained.
Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, CT, USA.
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Chen
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Troiano
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Kacena
D000818:Animals; D001842:Bone and Bones / Q000201:enzymology* / Q000378:metabolism; D002356:Cartilage / Q000201:enzymology* / Q000378:metabolism; D002863:Chromogenic Compounds / Q000737:chemistry; D005697:Galactosides / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D008689:Methacrylates / Q000737:chemistry; D051379:Mice; D008822:Mice, Transgenic; D008867:Microtomy / Q000379:methods; D044162:Parathyroid Hormone-Related Protein / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D016610:Tissue Embedding / Q000379:methods; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D002863:Chromogenic Compounds; D005697:Galactosides; D007211:Indoles; D008689:Methacrylates; D044162:Parathyroid Hormone-Related Protein; C005044:hydroxyethyl methacrylate; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside