Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA.
Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20 degrees C were examined for human promyelocytic leukemia cells (HL-60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti-BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double-stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10-20 min at 20 degrees C allowed denaturation of more than 80% of DNA and the maximum BrdUrd-linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.
Sasaki
K
K
Department of Pathology, Yamaguchi University School of Medicine, Ube, Japan.
Adachi
S
S
Yamamoto
T
T
Murakami
T
T
Tanaka
K
K
Takahashi
M
M
D001973:Bromodeoxyuridine / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D004273:DNA, Neoplasm / Q000032:analysis / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D006851:Hydrochloric Acid / Q000494:pharmacology*; D007150:Immunohistochemistry; D007938:Leukemia / Q000473:pathology; D009691:Nucleic Acid Denaturation / Q000187:drug effects*; D011419:Propidium / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
D004273:DNA, Neoplasm; D011419:Propidium; D001973:Bromodeoxyuridine; D006851:Hydrochloric Acid