Hydrochloric acid denaturation of colorectal tumour tissue infiltrated with bromodeoxyuridine.
The thymidine analogue, bromodeoxyuridine, is substituted into DNA during DNA synthesis and can be used to identify those cells which have passed through the S-phase of the cell cycle. Incorporated bromodeoxyuridine can be detected using anti-bromodeoxyuridine monoclonal antibodies which bind to the exposed bromodeoxyuridine in single-stranded DNA after a suitable denaturation step. Hydrochloric acid is the most commonly employed denaturation agent in bromodeoxyuridine monoclonal antibody methodologies. This preliminary study was to validate the hydrochloric acid denaturation step for colorectal tumour tissue infiltrated in vivo with bromodeoxyuridine. Standard immunohistochemistry and flow cytometric techniques using Bu20a were employed across a range of hydrochloric acid concentrations. Although high labelling indices were achieved using acid concentrations of 0.10 M HCl, the optimal hydrochloric acid concentration was not the same in all tumours. Sensitivity of DNA to hydrochloric acid denaturation should be carefully considered in bromodeoxyuridine methodologies using the monoclonal antibody Bu20a.
Williamson
K
K
Department of Surgery, Queen's University of Belfast, Northern Ireland.
Gilliland
R
R
Weir
H
H
Grimes
J
J
Hamilton
P
P
Anderson
N
N
Crockard
A
A
Rowlands
B
B
D000911:Antibodies, Monoclonal / Q000276:immunology; D016477:Artifacts; D001973:Bromodeoxyuridine / Q000032:analysis*; D002454:Cell Differentiation; D015179:Colorectal Neoplasms / Q000737:chemistry / Q000473:pathology*; D004261:DNA Replication; D004273:DNA, Neoplasm / Q000032:analysis / Q000187:drug effects*; D004277:DNA, Single-Stranded / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D005629:Frozen Sections; D006801:Humans; D006851:Hydrochloric Acid / Q000494:pharmacology*; D007150:Immunohistochemistry; D009691:Nucleic Acid Denaturation; D010434:Pepsin A; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D010434:Pepsin A; D001973:Bromodeoxyuridine; D006851:Hydrochloric Acid