1. The collected
CHIFF was resuspended in TEBP at a density of 2 x 109 cell
equivalents (ceq)/ml. BSA and NaCl were added to 1mg/ml and 150mM,
2. Preabsorbed cAR1
antiserum was added at 1:200 dilution. Mixing was carried out for 1-2 hours at
3. After 3 washes in
TEB plus NaCl and BSA, the sample was combined with 1:25 or 1:4 (for
saturation staining) diluted gold-conjugated secondary antibody (10nm size,
Amersham) in TEB plus NaCl and BSA and incubated at room temperature for 1-2
hours. Five washes were carried out in TEB.
Follow EM facility
4. The final pellet
was fixed with fresh 2% glutaraldehyde in 50 mM cacodylate buffer
pH 6.8 (22o for 30 minutes), postfixed with 1% OsO4 (30
minutes on ice), and stained with EM facility stocks of uranyl acetate and
5. Wash 5x in dH20
and then dehydrate in at least one hour 50%, 70%, 90%, then 100% ethanol
overnight. Wash 3x at least one hour with propylene oxide and switch to
6. Move to capsule
and embed in Eponate (Pella). At least one hour each 50% Ep:PO, 70%
Ep:PO, 90% Ep:PO, then 100% Ep overnight. Bake overnight thin sections were
7. Examined under a
Philips 410 TEM. Control samples were prepared in parallel with preimmune
serum to insure that the observed gold labelling was specifically dependent on
the interaction of cAR1 with anti-cAR1 antibodies.
1. Cells were
plated onto 35 mm petri dishes and then fixed/permeabilized with 2%
glutaraldehyde in 50 mM cacodylate buffer containing 1% CHAPS for 5 minutes.
Inclusion of CHAPS
was necessary to permeabilize the cells in order for antibody molecules to
access the C-terminus of cAR1.
2. Cells were
then postfixed in 0.5% glutaraldehyde for 10-15 minutes.
3. The fixed
cells were washed 2 x 15 minutes with 1 mg/ml NaBH4 in water to
quench glutaraldehyde, and then blocked for 30 minutes with 3% BSA in wash
Affinity-purified cAR1 antibody (1:200 dilution) was added and incubated for 1
hour. 5) After three 5 minutes washes with wash buffer, 1:20 diluted secondary
antibody-gold conjugate (5nm size, Amersham) was added and further incubated
for 1 hour.
After six 5 minutes
washes, cells were further fixed with 1% OsO4.
Samples were then
dehydrated, embedded in Eponate resin (Ted Pella), sectioned and post-stained
with 1% uranyl acetate.