Nov 12, 2023
Electron microscopy (EM) and quantification
- 1University of California, San Diego
Protocol Citation: Leonardo A Parra-Rivas 2023. Electron microscopy (EM) and quantification. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3zm6l3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 12, 2023
Last Modified: November 12, 2023
Protocol Integer ID: 90812
Abstract
Electron microscopy (EM) and quantification
Electron microscopy (EM)
Electron microscopy (EM)
Hippocampal neurons were plated at a density of
60,000 cells/cm2. DIV-3 neurons were infected with lentiviruses
carrying α-syn tagged at the C-terminus to mScarlet, or mScarlet alone
(multiplicity of infection or MOI=2.5). The transduced neurons were cultured to
maturity (DIV17-DIV21) before imaging. The ~100 % infection efficiency of the
mScarlet-tagged constructs was confirmed before using the coverslip for EM
experiments.
EM embedding protocol for monolayer cultured cells was performed
on ice. The coverslips were fixed in 2 % glutaraldehyde and in 0.1 M sodium
cacodylate (SC buffer) buffer for at least 60min at 4 °C, washed 3 min with 0.1
M SC buffer (5 times), and postfixed in 1% osmium tetroxide (OsO4) diluted in
0.1 M SC buffer for 30 min to 1hr.
The coverslips were then washed for 3 min (5
times) with 0.1 M SC buffer, followed by a water rinse, sequential ethanol
dehydration (20%, 50%, 70%, 90%, 2x100%) 1min each, and subsequent incubation
with dry acetone for 1 min at room temperature (2 times).
The samples were then incubated with a Durcupan:Acetone solution (50:50%) for 1hr in rotation,
followed by incubation with 100% Durcupan for 1 hr (2 times). The cells were
then embedded using foil plates along with wood sticks in the embedding oven
(overnight incubation).
60 nm ultrathin sections were cut with a diamond knife
and mounted on 300 mesh grids. Grids were post-stained with 2% of uranyl
acetate for 5 min and lead for 1 min. JEOL 1400 Electron Microscopy and Gatan
digital Camera were used to obtain images.
quantification
quantification
For quantification of inter-vesicular (IV) distances between vesicles, we
considered all the vesicles in the synapse encompassing 1µmx1µm. To mark each
vesicle, a line was drawn across the center of the vesicle, recording its
diameter and its position.
These parameters were then fed into our algorithm on MATLAB (RRID:SCR_001622)( http://www.mathworks.com/products/matlab/), which gave the output of IV distances
for a particular image. The IV distance for all images per condition was pooled
together. This algorithm is available upon request.