-transparent.png){kind=logo}
A new procedure is described to generate single-stranded DNA by exonuclease III (Exo III) digestion for bromodeoxyuridine (BrdU) immunocytochemistry on tissue sections. We compared this procedure with the most widely used procedure of DNA denaturation with 2 N HCl. In vivo and in vitro pulse and continuous labelling of tissues and cells were used. The specimens were fixed in formalin, ethanol, glutaraldehyde, Carnoy's, Bouin's or Zamboni's fixative and embedded in paraffin or used unfixed as cryostat sections or cytospin preparations. After Exo III digestion, BrdU substituted DNA was detected irrespective of the fixation procedure applied. The optimal protocol for nuclease digestion appeared to be simultaneous incubation, of 10 Units Exo III per ml EcoRI buffer and anti-BrdU monoclonal antibody at 37 degrees C. The advantages of Exo III digestion for BrdU immunocytochemistry compared to acid denaturation were: less non-specific nuclear background reactivity, no DNA renaturation, less DNA loss, optimal nuclear morphology, increase in antibody efficiency and the possibility for simultaneous detection of acid-sensitive tissue constituents. Disadvantages of the Exo III digestion are decreased sensitivity and the need for more rigorous pepsin pretreatment. We conclude that Exo III digestion of DNA is an appropriate alternative for acid denaturation for BrdU immunocytochemistry on sections of pulse-labelled specimens.
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
February 1976,
Biochemistry,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
January 1979,
Virology,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
July 1997,
Annales de pathologie,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
January 1987,
Methods in enzymology,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
August 2008,
CSH protocols,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
January 2008,
Methods in molecular biology (Clifton, N.J.),
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
January 2002,
Methods in molecular biology (Clifton, N.J.),
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
June 1984,
Gene,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
July 1999,
Cytometry,
W N Dinjens, and
J ten Kate, and
M H Lenders, and
E P van der Linden, and
F T Bosman
January 1991,
Growth factors (Chur, Switzerland),
