Expression of Collagen I, Smooth Muscle a-Actin, and Vimentin ...

3322 Investigative Ophthalmology & Visual Science, November 1993, Vol. 34, No. 12
and stored at — 70°C until use or were subjected to in
situ hybridization as described below.
Extraction of Total RNA
All solutions were autoclaved in the presence of 0.1%
diethylpyrocarbonate (DEPC), and all glassware and
appliances were either baked overnight or soaked in 1
M NaOH for 2 hours. Total RNA was isolated from
alkali-burned rabbit corneas and normal rabbit corneas
with RNAzol (Molecular Research Center, Inc.,
Cincinnati, OH), as previously described. 21 The RNAs
were resuspended in a solution containing 0.5% SDS
and 0.1 U/ml RNasin (Promega, Madison, WI) and
stored at —70°C until use.
Slot-Blot Hybridization
About 20 fig of total RNA from each sample were
denatured in 50% formamide at 65°C for 5 minutes
and were twofold serially diluted. The RNA was then
transblotted to BAS membranes with a slot-blotter
(Schleicher & Schuell) 21 and hybridized with the 32 P-
labeled cDNA of a 1(1) mRNA, as described previously.
22
In Situ Hybridization
The normal, alkali-burned, and lacerated rabbit corneas
were fixedin 4% methanol-free EM-grade formaldehyde
(Polysciences, Inc., Warrington, PA) in 0.05 M
sodium PBS, pH 7.4, at 4°C for 1 hour, and then
immersed in 30% sucrose in DEPC-treated water for 2
hours at 4°C. The specimens were embedded in OCT
compound (Miles, Elkhart, IN), quick frozen in ethanol/dry
ice, and stored at -70°C. Frozen sections of
5 fim thickness were cut by a cryostat and mounted on
Superfrost/Plus microscope slides (Fisher Scientific,
Pittsburgh, PA). The sections were then subjected to
hybridization with 3 H-labeled cDNA probes of rabbit
al(I) mRNA, as previously described. 21
A 3 H-labeled DNA probe was prepared by [ 3 H]-
dCTP incorporation using random primers as previously
described. 21 The specificity of the probe was 2 X
10 7 cpm/fxg. The hybridization reaction was carried
out at 45°C overnight. The sections were washed
under stringent conditions to reduce the background,
as previously described. 21 The slides were then dehydrated
in graded ethanol, air dried, and dipped in 50%
Kodak NT-2 Nuclear emulsion (Eastman-Kodak, Manchester,
NY); after exposure for 2 to 4 weeks, the
slides were developed for 3 minutes in a Kodak D-19
developer at 15°C, counterstained with hematoxylin
and eosin, and mounted.
Immunohistochemistry
For detection of collagen I, frozen sections of 5 /um
thickness were prepared from the OCT-embedded,
unfixed rabbit corneas by a cryostat and mounted on
Superfrost/Plus microscope slides (Fisher Scientific).
Cryosections were subjected to immunostaining using
the ABC method (avidin-biotin-peroxidase complex),
described by Chida et al. 23 For detection of a-SMA,
vimentin, and desmin, sections (3 /xm) of paraffin-embedded
corneas were mounted on precleaned glass
slides without adhesive. The sections were deparaffinized
and rehydrated with PBS before incubation with
antibodies.
Briefly, the tissue sections were incubated with
0.3% H 2 0 2 in methanol at room temperature for 30
minutes to eliminate endogenous peroxidase activity
in tissue. The sections were rinsed with PBS and incubated
at room temperature for 120 minutes with
preimmune serum of the source of biotinylated second
antibody (10X diluted) to block nonspecific absorption
of second antibodies to the tissue sections.
The sections were incubated with goat polyclonal anticollagen
I antibodies (1 jug/ml) or appropriately diluted
monoclonal antibodies (0.1 to 1 jig/ml) at room
temperature for 60 minutes or 4°C overnight. The
sections were washed with PBS and further incubated
with biotinylated second antibody for 60 minutes at
room temperature, followed by incubation with streptavidin-biotin-peroxidase
complex at room temperature
for 60 minutes. The sections were soaked in a
solution containing 0.2 mg/ml of 3-3'-diaminobenzidine
hydrochloride, 0.005% of H 2 0 2 , and 50 mM Tris-
HC1 buffer, pH 7.6, for 3 to 5 minutes and counterstained
with Mayer's hematoxylin. Photomicrograms
were prepared with a Nikon Diaphot microscope (Nikon,
Garden City, NY).
RESULTS
Slot-Blot Hybridization With 32 P-Labeled
cDNAofal(I)mRNA
Total RNA was extracted from alkali-burned rabbit
corneas that had healed for 1, 2, 3, 5, 7, 14, and 21
days with RNAzol, and it was subjected to slot-blot
hybridization with 32 P-labeled cDNA of a 1(1) mRNA
as previously described. 21 Figure 1 demonstrates that
levels of a 1(1) mRNA steadily increase and reach a
plateau as the injured corneas are allowed to heal for
more than 14 days. These results indicate that the alkali-burned
corneas actively synthesize collagen I in
attempts to repair the destroyed extracellular matrix
induced by the alkali treatment.
In Situ Hybridization With 3 H-Labeled cDNA
Probe of al (I) mRNA
To identify the cells synthesizing collagen I in alkaliburned
corneas, the injured corneas that had healed
for 1 day and for 1,2, and 3 weeks were subjected to in