Muller Cell Expression of Gliol Fibrillory Acidic Protein offer Genetic ...

1322 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / November 1984 Vol. 25
each containing two 15-watt fluorescent bulbs were
positioned 18 cm above the bottom of the cage.
These conditions resulted in an incident luminance
of approximately 200-ft candles at the floor of the
cage. The temperature in the cage was 24 ± 1 °C.
Age-matched Sprague-Dawley rats maintained in a
12-hr overhead room light/ 12-hr dark environment,
were used as controls.
All rats were enucleated under ether anesthesia
between 1:00 and 2:30 PM. After a slit was made in
the cornea, the lens and vitreous were removed and
the globe was immersed in 4% formalin in 0.13 M
phosphate buffer (pH 7.4).
Immunofluorescence
After 6 hr in fixative at room temperature, the
eyes were bisected, transferred to 30% sucrose in 0.13
M phosphate buffer and stored at 4°C overnight.
Sections were cut at a thickness of 20 nm using a
cryostat at —20°C. The sections were mounted on
chrome alum-gelatin coated slides and air-dried overnight
at room temperature. Plastic rings (0.75-cm
diameter) were mounted with fingernail polish around
the sections to form incubation wells. Each well
contained an experimental section of retina (RCS or
CL-damaged) and a control section. The sections
were treated for 10 min at room temperature with
1% goat serum and 4% bovine serum albumin (BSA)
in phosphate buffered saline (PBS) followed by overnight
incubation at 4°C in GFAP antiserum diluted
1:100 in PBS containing 0.3% Triton X-100. The
GFAP antiserum, provided by Dr. Larry Eng (Veterans
Medical Center; Palo Alto, CA), was raised in
rabbits against GFAP obtained from multiple sclerosis
plaques. 8 Control sections were treated identically
with an IgG fraction from preimmune rabbit serum.
Sections were washed twice (15 min each) with PBS
at room temperature and incubated for 30 min at
room temperature in the dark in sheep anti-rabbit
IgG-fluorescein isothiocyanate (Cappel Laboratories),
diluted 1:50 in PBS with 0.3% Triton X-100. After
two 10-min washes in phosphate buffer, the plastic
rings were removed and the sections were coverslipped
with 80% glycerol in 0.13 M phosphate buffer containing
5% n-propyl gallate. 9 The sections were examined
with a Zeiss microscope equipped for epifluorescence.
Measurement of Outer Nuclear Layer
After enucleation, eyes were stored in fixative at
4°C overnight. They were bisected vertically just
temporal to the optic nerve head. The bisected eyes
were washed for several hours in phosphate buffer
and then dehydrated through a graded series of
ethanol. The half of the eye including the optic nerve
head was embedded in plastic (Sorvall Embedding
Medium), with the cut edge of the eyecup placed flat
to allow for sectioning along the inferior-superior
plane, including the optic nerve head. In some cases
the eyes were bisected after 6 hr in fixative, and the
half without the optic nerve head was processed for
immunofluorescence, while the remaining half was
stored in fixative overnight.
Sections were cut at a thickness of 2.5 ^m on a
Sorvall JB-4 microtome and stained for 30 sec with
10% Richardson's stain. A section through the optic
nerve head from each eye was chosen and the thickness
of the outer nuclear layer was measured 250
ixm, 500 fim, and 750 /xm from the optic nerve head
in the superior and inferior hemispheres. The mean
and standard error of the mean of these six measurements
were calculated.
Preparation of Triton X-100 Insoluble Proteins
Triton-insoluble proteins were obtained from retina
according to Pruss et al. 10 Four to six retinas were
homogenized in ice-cold PBS containing the protease
inhibitors p-chloromercuribenzoate, phenyl methane
sulfonyl fluoride and o-phenanthraline, each at 1
raM concentration. The homogenate was centrifuged
at 8000 g for 10 min at 4°C. The pellet was extracted
with PBS containing 0.6 M KC1, 0.5% Triton X-100
and the protease inhibitors, and centrifuged at 8000
g for 10 min at 4°C. After a second extraction with
Triton X-100, the pellet was washed four times in
ice-cold PBS and solubilized by boiling in SDSpolyacrylamide
sample buffer." In the light damage
experiments, Sprague-Dawley rats had been exposed
to constant light for 3 or 7 days. Control animals
had been kept in 12-hr light/12-hr dark cycle. RCS
rats were 45 and 70 days old. Congenic, 45-day-old
^y" 1 " rats were used as controls.
Polyacrylamide Gel Electrophoresis and
Electroblotting to Nitrocellulose
One dimensional SDS-polyacrylamide gel electrophoresis
(PAGE) was performed according to the
procedure of Fairbanks et al" using protein standards
ranging in molecular weight from 15-94,000. Proteins
were transferred from PAGE gels to nitrocellulose
membranes (BIORAD) in a Hoefer Transphor electrophoresis
apparatus at 0.8 mV for 1 hr at room
temperature. 12 After 1 hr blocking in Tris-buffered
saline (TBS) containing 3% BSA, the blots were
treated overnight in anti-GFAP diluted in TBS and
1% BSA. After several washes, the blots were incubated
with goat anti-rabbit IgG (Cappel) for 1 hr (1:1000
dilution in TBS and 1% BSA). Following a 30-min
exposure to the peroxidase-antiperoxidase complex