Biology Reference
In-Depth Information
if discrimination of individual cells is critical for the interpretation of the data, it
would be better to grow them to a subconfluent density.
21.5.3
Colloidal gold conjugates
We have almost exclusively used colloidal gold reagents with a gold particle size of
10 nm. Gold particles of 5 nm are more difficult to detect unambiguously due to the
often irregular surface and high contrast of the replicas. Use preferably freshly pur-
chased gold conjugates as long-term storage might increase aggregation of the re-
agent. The aggregation state of the gold reagent can be tested by adsorbing
diluted suspensions of gold conjugates onto collodion/carbon-coated EM grids
and analyzing in TEM.
21.5.4
Cell surface replication and replication of fracture surfaces
Upon attachment to a support lymphocytes will, in contrast to adherent cells, not form a
continuous monolayer. This discontinuity makes it more difficult to detach the metal
replica fromthemica sheet. Covering the cellswith anothermica sheet, thereby creating
a mica sandwich, allows formation of a continuous thin layer of buffer that afterward
facilitates the generation of a continuous replica that can be floated in the cleansing
solution. When you split the frozen mica sandwich, most cells remain attached to the
poly-
L
-lysine-coatedmica and will be surface replicated, withmost of the gold particles
stabilized by the replica. Some cells can be fractured by the splitting of the mica sand-
wich. If the cell is fractured through the lipid bilayer of the nonadhered membrane, the
gold label will be lost, even though the replica has a very similar appearance to nonfrac-
tured cells. On the other hand, if the fracture plane follows the plasma membrane
attached to the mica surface, one will not observe the bulge caused by the nuclear area,
but the gold particles will remain attached to the exoplasmic hemimembrane under the
replica and can be visualized and quantified in the TEM. It should however be taken into
account that the membrane represented by this replica was in contact with the mica
support, which could give rise to a change in distribution of the cell surface molecules
studied as compared to the nonadhered membrane.
21.5.5
Primary antibody and fixation conditions
If primary antibodies do not work under the fixation conditions used, one could try to
lower the concentrationofPFAto1%or omit theprefixation step.However, cells should
then be kept all the time on ice and could be labeled in presence of 0.02% of NaN
3
,in
order to reduce the risk of antibody-induced capping of the cell surface receptor.
21.5.6
Secondary reagents
Dilutions of the colloidal gold-coupled secondary reagent can be tested on a batch of
cells stained with saturating concentrations of the primary antibody (tested by flow
cytometry), followed by replica generation and analysis. Use an isotype control
