An
EcoRI/
XbaI fragment of CPV2 containing
the entire 877-bp SV40 intron and polyadenylation sequence was
subcloned into Bluescript KS
− (Stratagene) to
generate a riboprobe vector as previously described.
12 Sense and antisense transcripts were produced by in vitro transcription
using 1 μg linearized riboprobe vector with 20 U of T3 (Stratagene)
and 20 U of T7 (Pharmacia, Piscataway, NJ) RNA polymerase,
respectively, for 3 hours at 30°C using
35S-labeled UTP as previously
described.
11 Hybridizations were performed on embryos
collected from timed pregnancies (morning of copulation plug = day
E0.5). The embryos were fixed in 4% paraformaldehyde, dehydrated, and
embedded in PARAPLAST X-TRA embedding wax (Oxford Labware, St. Louis,
MO). Tissue sections (5 μm) were collected on Superfrost Plus
microscope slides (Fisher Scientific, Fair Lawn, NJ), deparaffinized in
xylenes, and rehydrated in a decreasing ethanol series. Sections were
treated with 0.2 N HCl for 15 minutes, rinsed in phosphate-buffered
saline and incubated in 20 μg/ml proteinase K (GIBCO/BRL, Rockville,
MD) in 50 mM Tris, pH 7.5; 5 mM EDTA for 7 minutes at 25°C. Slides
were rinsed in 4% glycine/phosphate-buffered saline and acetylated
with 0.25% acetic anhydride made in 0.2 M triethanolamine–HCl, pH
8.0. Hybridizations were carried out overnight at 50°C in 0.3 M
NaCl/10 mM Tris–HCl, pH 7.4/10 mM
NaH
2PO
4/5 mM EDTA/0.2%
Ficoll 400/0.2% polyvinyl pyrrolidone/50 mM dithiothreitol/0.5 mg/ml
polyadenylic acid/50 mg/ml yeast tRNA/10% dextran sulfate/50%
formamide/0.25 mM γ-S-thio ATP (Sigma Chemical, St. Louis, MO).
Approximately 20 ng of
35S-labeled sense or
antisense riboprobe/slide was added to the hybridization mixture.
Slides were washed in: FSM (50% formamide/0.3 M NaCl/30 mM citric
acid, pH 8.0/20 mM β-mercaptoethanol) at 65°C twice for 30 minutes;
STE (0.6 M NaCl/60 mM citric acid, pH 8.0/20 mM Tris–HCl, pH 7.4/1 mM
EDTA) at 37°C twice for 10 minutes; STE supplemented with 6 μg/ml
RNase A at 37°C for 30 minutes; STE supplemented with 20 mMβ
-mercaptoethanol at 37°C for 10 minutes; FSM at 65°C twice for
45 minutes; 0.3 M NaCl/30 mM citric acid, pH 8.0 at 37°C for 10
minutes; and 15 mM NaCl/1.5 mM citric acid, pH 8.0 at 25°C for 5
minutes. Hybridized slides were air-dried, dipped in Kodak NTB-2
emulsion, and exposed for 4 days at 4°C before being developed with
Kodak D-19 developer. Slides were counterstained with hematoxylin and
eosin.