TEM imaging
was performed
using an FEI Tecnai 12 Spirit BioTWIN, operated at 100 kV with a 2
× 2 k Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH,
Münster, Germany). 10–15 images were taken randomly
for each sample at different magnifications between 20 000×
and 60 000×. Five microliters of sample was spotted on
a 400 mesh Formvar/carbon-coated copper grid and incubated for 10
min. The grid was then washed twice with 5 μL of Milli-Q (MQ)
water, stained with 5 μL of 1% uranyl formate for 5 min, and
air-dried. For immuno-EM, 5 μL samples were spotted on the 200-mesh
nickel grid. After 10 min, the excess sample was blotted with Whatman
filter paper. The grid was blocked by using 5 μL of 1% bovine
serum albumin (prepared in PBS). After 30 min, the grid was washed
thrice with MQ water. Then, 10 μL of primary mouse antibody
(6E10, 1:200 in PBST, BioLegend) was dropped on the grid and incubated
at room temperature for 60 min. It was then washed thrice with PBST
followed by incubation with secondary antibody (antimouse IgG-gold,
1:40 dilution in PBST, BBI Solutions, Crumlin, UK) for 60 min. The
grid was then washed three times with PBST and then stained as described
above.
was performed
using an FEI Tecnai 12 Spirit BioTWIN, operated at 100 kV with a 2
× 2 k Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH,
Münster, Germany). 10–15 images were taken randomly
for each sample at different magnifications between 20 000×
and 60 000×. Five microliters of sample was spotted on
a 400 mesh Formvar/carbon-coated copper grid and incubated for 10
min. The grid was then washed twice with 5 μL of Milli-Q (MQ)
water, stained with 5 μL of 1% uranyl formate for 5 min, and
air-dried. For immuno-EM, 5 μL samples were spotted on the 200-mesh
nickel grid. After 10 min, the excess sample was blotted with Whatman
filter paper. The grid was blocked by using 5 μL of 1% bovine
serum albumin (prepared in PBS). After 30 min, the grid was washed
thrice with MQ water. Then, 10 μL of primary mouse antibody
(6E10, 1:200 in PBST, BioLegend) was dropped on the grid and incubated
at room temperature for 60 min. It was then washed thrice with PBST
followed by incubation with secondary antibody (antimouse IgG-gold,
1:40 dilution in PBST, BBI Solutions, Crumlin, UK) for 60 min. The
grid was then washed three times with PBST and then stained as described
above.