Immunohistological optimization of detection of bromodeoxyuridine-labeled cells in decalcified tissue.
Mice were injected with a range of bromodeoxyuridine (BrdU) concentrations from 0.01 mg to 10 mg, and their jaws were fixed in buffered formalin or modified Carnoy. After EDTA or formic acid decalcification, a range of DNA denaturation schedules was assessed and immunohistological detection of BrdU-containing nuclei was performed using the Sera Lab anti-BrdU antibody MAS 250b. For Carnoy-fixed tissue, denaturation in 1 N HCl for 8 min at 60 degrees C was capable of adequately detecting an injected dose of 0.05 mg but not a dose of 0.01 mg BrdU, whereas pepsin/HCl treatment gave only weak staining after injection of 1 mg BrdU. In comparison, formalin fixation required pre-treatment with 0.2-0.4% pepsin/HCl at 37 degrees C for comparable staining intensity, but could still not adequately detect a dose of 0.1 mg BrdU. There was little detectable difference in staining between EDTA- and formic acid-decalcified tissues after injection of 10 mg BrdU.
Hume
W J
WJ
Department of Dental Surgery, Leeds University Dental School, United Kingdom.
Keat
S
S
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism*; D001854:Bone Marrow Cells; D001973:Bromodeoxyuridine / Q000378:metabolism*; D003648:Decalcification Technique; D004492:Edetic Acid; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D005561:Formates; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D008334:Mandible / Q000166:cytology / Q000378:metabolism; D051379:Mice; D014070:Tooth / Q000166:cytology / Q000378:metabolism*
D005561:Formates; D004492:Edetic Acid; D001973:Bromodeoxyuridine