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Brdu
5’-bromo-2’-deoxyuridine
(BrdU) is a thymidine analogue that incorporates into dividing
cells during DNA synthesis during S phase of the cell cycle
(Gratzner, 1982). BrdU will replace thymidine during replication
and once incorporated into the new DNA, it will remain in
place and will be passed down to its progeny upon division.
Specific antibodies designed to bind BrdU, and conjugated
with fluorochromes, are used to label BrdU positive cells.
The
development of BrdU immunohistochemistry to identify S-phase
cells in the brain (Miller and Nowakowski, 1988) was an
important advance over 3H thymidine autoradiography. BrdU
method can be used to detect labelled cells throughout the
thick tissue sections required by stereologic techniques
for determining the total number of S-phase cells within
a brain region (Gratzner, 1982).
The
method consists of injection of BrdU into the intraperitoneal
cavity followed by a variable survival time allowing for
tracking the fate of divided cells and their progeny. At
the end of the experiment, the animal is sacrificed and
the tissues fixed with a standard paraformaldehyde-based
fixative. BrdU is detected in the tissue using specific
primary antibodies.
The
substitution of an endogenous DNA base, Thymidine, with
the BrdU analogue ensures specific labelling of only the
dividing cells. This specificity of BrdU combined with the
high sensitivity of fluorescence microscopy; including confocal
microscopy has led to the great popularity of the method.
One
useful feature of BrdU is its long-term retention in divided
cells and its passage to their daughter cells. This feature
can be used to trace the cell lineage and cell survival.
A spectacular example of such use was a study by Eriksson
and colleagues who injected patients with BrdU and then
examined brain tissue post-mortem up to 2 years later.
However,
BrdU also has some drawbacks. First, it may not be absolutely
specific for dividing cells, i.e., BrdU could potentially
be picked up by cells that are undergoing repair of DNA
in addition to cells replicating their complete genome (Korr
et al., 1989; Selden et al., 1993; Schmitz et al., 1999).
Additionally,
BrdU may act as a mutagen in cells that have picked up BrdU.
High doses of BrdU have also been shown to have adverse
effects, consequently resulting in severe abnormalities
of the developing tissues on embryonic and neonatal rats
(Bannigan, 1985; Kolb et al., 1999).
These
side effects aside, the use of BrdU in whole-animal experiments
is difficult due to uncertainty of diffusion of this substance
among the various tissues of the body following intraperitoneal
injection. For example, the blood brain barrier may prevent
the BrdU from freely penetrating the brain tissue, especially
in old animals (Cameron and McKay, 2001).
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