To visualize single C1q protein particles by TEM, C1q (5 μg/ml)
was diluted in PBS. In order to gold-label ApoE, biotinylated plasma-purified
ApoE or ApoE139-152 (20 μg/ml) was incubated with
streptavidin-gold complexes (5 nm gold, British BioCell International Ltd.)
diluted 1:25 in PBS for two hours at RT. C1q (10 μg/ml) was added to the
ApoE-streptavidin-gold solution (1:1 mixture) and incubated under gentle shaking
for two hours at RT. To detect single C1q-ApoE3 complexes by structure, full
length ApoE3 (40 μg/ml) was directly labeled with EM-Grade 6 nm gold
particles (AURION-ImmunoGold Reagents & Accessories, The Netherlands).
The probe (containing ~2 x 1014 gold particles/ml) was diluted
1:200 in PBS. Carbon-coated grids were hydrophilized by glow discharge at low
pressure in air. Aliquots of C1q alone and C1q-ApoE-streptavidin-gold or
C1q-ApoE3-gold were adsorbed onto hydrophilic, carbon-coated grids for 1 min,
washed twice with ddH2O, and stained on a drop of 2% uranyl acetate
in ddH2O. Specimens were analyzed with a Zeiss EM902A electron
microscope (Carl Zeiss) operated at 80 kV accelerating voltage, and images were
recorded with a FastScan-CCD-camera 1,024 x 1,024 (TVIPS).
was diluted in PBS. In order to gold-label ApoE, biotinylated plasma-purified
ApoE or ApoE139-152 (20 μg/ml) was incubated with
streptavidin-gold complexes (5 nm gold, British BioCell International Ltd.)
diluted 1:25 in PBS for two hours at RT. C1q (10 μg/ml) was added to the
ApoE-streptavidin-gold solution (1:1 mixture) and incubated under gentle shaking
for two hours at RT. To detect single C1q-ApoE3 complexes by structure, full
length ApoE3 (40 μg/ml) was directly labeled with EM-Grade 6 nm gold
particles (AURION-ImmunoGold Reagents & Accessories, The Netherlands).
The probe (containing ~2 x 1014 gold particles/ml) was diluted
1:200 in PBS. Carbon-coated grids were hydrophilized by glow discharge at low
pressure in air. Aliquots of C1q alone and C1q-ApoE-streptavidin-gold or
C1q-ApoE3-gold were adsorbed onto hydrophilic, carbon-coated grids for 1 min,
washed twice with ddH2O, and stained on a drop of 2% uranyl acetate
in ddH2O. Specimens were analyzed with a Zeiss EM902A electron
microscope (Carl Zeiss) operated at 80 kV accelerating voltage, and images were
recorded with a FastScan-CCD-camera 1,024 x 1,024 (TVIPS).