Five-week-old tubby and wild-type matched control animals were
anesthetized with tribromoethanol and perfused with phosphate-buffered
saline (PBS) followed by 4% paraformaldehyde in PBS. Eyes were
enucleated and postfixed in the same fixative for 18 hours, dehydrated,
and embedded in paraffin. A minimum of three eyes from three different
animals in each group were used for this experiment. Sections of 6-μm
thickness were cut and mounted on slides pretreated with poly l-lysine (Sigma, St. Louis, MO). Sections were
deparaffinized in xylene and rehydrated through a graded series of
alcohol and PBS. Sections were then treated with proteinase K (20μ
g/ml) for 7 minutes at room temperature (RT), acetylated in 0.1 M
triethenolamine (pH 8.0)-0.25% acetic anhydride solution, dehydrated,
and air dried.
[α-
33P] UTP-labeled sense and antisense
riboprobes (10
5 cpm/μl) were generated from
plasmids containing cDNA fragments of
tub (nt 197–599,
U52433),
Tulp1 (nt 81–707, AF105,711),
Tulp2 (nt
40–1011, AF105,712), and
Tulp3 (nt 44–623, AF045,583). To
prevent cross-hybridization, all probes were prepared from the
amino-terminal half of each protein, where the sequence homology
between family members is low.
6 7
To confirm the specificity of these probes, northern hybridization was
performed as described in Nishina et al.
7 Membranes with 5μ
g poly(A)
+ RNA from mouse eyes and testes were
hybridized with
32P random-labeled DNA probes for
tub,
Tulp1, and
Tulp2 obtained from
the clones described above. The specificity for the
Tulp3 probe has previously been confirmed.
7
Hybridization solution (50% formamide, 0.3 M NaCl, 20 mM Tris-HCl [pH
8.0], 5 mM EDTA, 10 mM NaPO4, [pH 8.0], 10%
dextran sulfate, 1× Denhardt’s, and 0.5 mg/ml yeast tRNA) containing
50,000 cpm/μl labeled probe was applied to each slide. Sections were
covered with coverslips and incubated at 60°C in a humidified chamber
overnight. The slides were washed in a solution of 50% formamide-2×
SSC at 65°C for 30 minutes, rinsed twice in NTE (0.5 M NaCl, 10 mM
Tris-HCl [pH 7.5], 5 mM EDTA) at 37°C for 15 minutes, treated with
RNaseA (1 μg/ml) at 37°C for 15 minutes, and rinsed in NTE for
another 15 minutes. The slides were then washed in a solution of 50%
formamide-2× SSC at 65°C for 20 minutes, in 2× SSC at RT for 15
minutes, and in 0.1× SSC at RT for 15 minutes and dehydrated. The
slides were coated with NTB-2 emulsion (Eastman Kodak, Rochester, NY)
and, after exposure for 5 days, 10 days, and 3 weeks (tub and Tulp1: 5 days, Tulp2: 3 weeks, Tulp3: 10 days) at 4°C, they were developed (D19; Eastman
Kodak) and counterstained with hematoxylin.