Biology Reference
In-Depth Information
antibody to verify the amount of background staining. For the 10 nm colloidal gold-
conjugated protein A, we have routinely used a threefold lower dilution than the one
recommended by the manufacturer (CMC Utrecht).
21.5.7
Freezing of samples
The cryogen container should be filled to the rim with secondary cryogen to avoid
precooling of the specimen in the gaseous layer of ethane that otherwise forms be-
tween the surface of the cryogen and the upper rim of the container. For the same
reason, one should only position the forceps injector, holding the tweezers and mica
sandwich, above the freezing chamber for the time needed to plunge the sample. Re-
move excess of the liquid ethane adhering to the mica with the filter paper before
transferring to the liquid nitrogen-filled cryotube. The ethane will freeze in liquid
nitrogen, making splitting of the mica sandwich just before loading on the specimen
table much more difficult.
21.5.8
Mounting of samples in the specimen table
Take care that the bench Dewar, specimen table, loading device, and tweezers are
clean and dry to avoid contamination of the sample and condensation of water onto
the sample, which can give rise to subsequent ice crystal formation. We store spec-
imen tables in a 56
C stove to assure that they are completely dry before use. Do not
breathe directly on the liquid nitrogen surface while mounting the micas on the table,
in order to avoid that the liquid nitrogen will reach its boiling point. This would give
rise to bubbles that impair visualization of the samples under the liquid nitrogen
surface.
21.5.9
Replica floating
If the replica does not detach from the mica upon submergence in the bleach solution,
one can scrape the edges of the mica with Dumont tweezers. Alternatively, use the
tweezers to scrape a grid pattern on the surface of the replica to obtain smaller pieces
of replica that may float more easily. One could in principle leave the mica sheet with
the replica still attached on the bottom of the well for digestion of the organic
material, but in our hands, floating of an unfolded replica piece is rare under these
conditions. An oily and inconsistent appearance of the replica indicates a too thin
carbon layer.
21.5.10
Replica stability
Extra support for the replicas can be provided by precoating the EM grids with a For-
mvar or carbon layer. This can be especially helpful when replicating small and
rounded naive T lymphocytes, which have a tendency to pop out of the replica. If
the replica pieces are tiny, they can be lost in the vacuum of the TEM column if
