Bromodeoxyuridine (BrdU) immunocytochemistry by exonuclease III (Exo III) digestion.

Publisher: Springer-Verlag Country of Publication: Germany NLM ID: 0411300 Publication Model: Print Cited Medium: Print ISSN: 0301-5564 (Print) Linking ISSN: 03015564 NLM ISO Abbreviation: Histochemistry Subsets: MEDLINE

Original Publication: Berlin ; New York : Springer-Verlag.

Bromodeoxyuridine/*analysis ; DNA/*metabolism ; DNA, Single-Stranded/*metabolism ; Exodeoxyribonucleases/*metabolism; Animals ; Antibodies ; Bromodeoxyuridine/immunology ; Immunohistochemistry ; Nucleic Acid Denaturation ; Rats ; Rats, Inbred Lew ; Sensitivity and Specificity

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.

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0 (Antibodies)
0 (DNA, Single-Stranded)
9007-49-2 (DNA)
EC 3.1.- (Exodeoxyribonucleases)
EC 3.1.11.2 (exodeoxyribonuclease III)
G34N38R2N1 (Bromodeoxyuridine)

Date Created: 19921001 Date Completed: 19930106 Latest Revision: 20190827

20221208

10.1007/BF00315878

1452452