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

Abstract

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.

Identification of DNA-Synthesizing Bacterial Cells in Coastal North Sea Plankton

“…Since heat (39), DNase (11,44), detergents (47), and HCl (2) are all known to be useful in recovering immunoreactivity of BrdU epitopes in formalin-fixed tissues and cell preparations, it was necessary to investigate the effects of a variety of agents in combination with each other and at different temperatures on BrdU cell-labeling indices (i.e., the percentages of hybridized cells that showed BrdU labeling). The best results (highest labeling indices) were achieved by using a combination of several permeabilizing and epitope-unmasking steps (Table 2).…”

“…Nuclease digestion was carried out simultaneously with antibody binding. This strategy has been employed previously (11,42) and is the recommended protocol in some commercially available cell proliferation assays (Boehringer Mannheim). For subsequent quantification of labeling indices of isolates and of DNA-synthesizing North Sea bacterioplankton populations by FISH and ICC, we used the optimal set of conditions determined in our preceding experiments (Table 2).…”

Simultaneous immunoenzymatic staining of catecholamines, catecholamine-biosynthesizing enzymes, and bromodeoxyuridine in adrenal medullary cells of the rat.

“…More recently, a procedure using treatment of sections with the enzyme nuclease was reported. However, if cross-linking fixatives such ,as formaldehyde are used, additional deproteinizing by pepsin is needed (Dinjens et al, 1992;Montuenga et al, 1992).…”

“…We have applied DNA denaturation with 2 N HC1 because this procedure is most effective and because other procedures require an even stronger pepsin pre-treatment (Dinjens et al, 1992).…”

Immunoelectron Microscopic Labeling of Digestive Tract Stem Cells by Means of Bromodeoxyuridine Antibody.

“…We think that sequential pretreatment is a necessary step for staining of Lowicryl K4M resin embedding materials. It is assumed that the etching of sections by hydrogen peroxide and the denaturation of nuclear proteins caused with hydrochloric acid treatments are favorable steps for immunocytochemical staining (3,6,11).…”