Biomedical Engineering Reference
In-Depth Information
to high background levels. Acetylation of the tissue is important for reducing
background signals (perhaps due to reducing the electrostatic binding of the probe
to basic [positively charged] amino groups on the brain section). It is critical that
the acetic anhydride is fresh and added to the TEA buffer just prior to use.
7. Prefi xation of tissue with Proteinase K, which can improve penetrance of the
probe, has not proven to enhance the signal in the freshly frozen brain sections
processed under the current fi xation procedure. However, when testing new
probes for which the hybridization signal is weak, experiments can be carried
out to determine whether or not the Proteinase K pretreatment (5-10 mg/L
of Proteinase K in 100 m
M
Tris, pH 8; 50 m
M
EDTA; 30 min at 37°C) can
improve the signal. Note that increasing the Proteinase K concentration will
damage the tissue, so test experiments should be carried out to determine the best
concentration for optimized hybridization signal with minimal tissue damage.
8. Pretreated brain sections can be stored (at least -30°C) for a number of days
(brain specimens have even been used up to 6 mo) prior to the hybridization
procedure without signifi cant loss of mRNA expression levels.
4.3. Probe Preparation
9. The size of the labeled probe can be determined on an acrylamide gel. High
specifi c labeling can result in smaller bands on the gels in addition to the major
band at the size of the cDNA insert. The synthesis of full-length riboprobes is
obtained mainly with low specifi c labeling in which unlabeled UTP is added
during the probe labeling. Riboprobes >1000 nucleotides can be used, but alkaline
hydroxylase (especially for those >1500 bases) is recommended after the probe
labeling to ensure adequate penetrance of the resulting shorter fragments.
10. It is always best to use freshly prepared labeled probes. However, labeled probes
(stored at -30°C) can be used a few days later (generally a week), but some
probe degradation can occur, resulting in increased background. When possible,
all reagents and chemicals including the isotopes should be stored frozen in
aliquots (to prevent freeze-thawing problems and to decrease the possibility
of contamination).
11. Although
35
S is the most common isotope used for riboprobe labeling,
33
P has
proven very effective for visualizing low expressing transcript (as well as those
with normal expression levels) with minimal background signal. For example,
opioid receptor mRNAs are normally much lower than the opioid neuropeptide
genes and the use of
33
P has proven much more effective for visualization of the
receptor mRNAs
(25)
. However, the half-life of
33
P is only 25 d, so this isotope
is not suitable when cellular visualization of very low expressing transcripts that
require long exposure of the emulsion-dipped slides.
12. The synthesis of CTP-labeled riboprobes works equally well for ISHH experi-
ments, and CTP- and UTP-labeled probes can even be combined when attempting
to increase the hybridization signal, for example, very low expressing transcripts.
However, increasing the concentration of the hybridization probes can also
increase background nonspecifi c signal, so it is important to determine the
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