Biomedical Engineering Reference
In-Depth Information
3.1. Tissue Preparation
For the coupling of
in situ
hybridization with retrograde tracing or immu-
nohistochemistry, the protocols described here need to be performed on fi xed
tissue.
1. Perfuse rat brains intracardiacally with 30 mL of 0.9% NaCl, then with 250 mL
of 1-4% PFA in 0.1
M
phosphate buffer.
2. Proceed for cryostat or vibratome sections as described in the following cor-
responding methodological sections.
3.2. Labeling of the Probes
All the probes are labeled using standard protocols as originally described
(10
,
14)
. Oligonucleotide probes are labeled with [
35
S]dATP by “tailing,” that
is, addition of nucleotides at the 3
end. Nick translation is used to label cDNA
probes by incorporation of the same radiolabeled deoxynucleotides. cRNA
probes are synthesized by in vitro transcription from cDNA clones using
[
35
S]UTP from 50 ng of linearized plasmid and with the appropriate RNA
polymerase (SP6, T7, or T3). For the choice of the probes and radiolabeled
nucleotides,
see
Note 1
.
3.3.
In Situ
Hybridization
Whatever the various protocols used,
in situ
hybridization is performed
with the three usual steps of pretreatment, hybridization of the probe, and
post-hybridization washes, with some specifi c steps depending on the type
of probe used.
3.3.1. Pretreatment
1. Thaw the slides for 10 min at room temperature.
2. Wash twice for 30 min in 4X SSC-0.1% Denhardt's solution (only for oligo-
nucleotides and cDNAs).
3. Wash the slides twice for 10 min in 4X SSC, then incubate for 10 min in 4X
SSC-1.33% triethanolamine-0.25% acetic anhydride, pH 8.0. This step is useful
to decrease the background by acetylation of free radicals.
4. Rinse twice 5 min in 4X SSC.
5. Dehydrate in graded ethanol (70%, 80%, 90%, 100%) and air-dry.
3.3.2. Hybridization
1. Microcentrifuge the labeled probe (usually stored in ethanol-acetate) for 1 h
at maximum speed.
2. Remove and check the supernatant, and dry the pellet under vaccum.
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