Biology Reference
In-Depth Information
repeatedly mounted in the TEM stage. It is therefore advisable to image all the rel-
evant cells of a single replica in one session and perform quantification off-line.
21.5.11
Replica quality
Replicas should have a smooth appearance. If they look pale and lack contrast, the Pt
deposition is insufficient. If the replica is too dark, there is an excess of Pt/C making
it difficult to discern gold particles.
21.5.12
Low labeling efficiency and implications
for interpretation of data
The total amount of gold particles we have quantified per cell indicated that we
obtained labeling efficiencies that reach around 5-15%. This has therefore not
allowed us to directly infer the distribution of the TCR or any other surface protein
from the distribution of the corresponding gold particles. However, the technique can
be used to compare receptor distribution between different populations of cells.
In the case of the TCR, we have obtained conformation of the validity of its com-
parative power by analyzing the TCR distribution of two T-cell populations by this
technique and by blue native PAGE (
Kumar et al., 2011
) and comparing methyl-
b
-
cyclodextrin-treated and nontreated cells (
Schamel et al., 2005
).
If the staining efficiency is calculated, one can get some information by Monte
Carlo simulations, in order to include or to exclude possible distributions of the
surface protein (
Fiala, Kaschek, Blumenthal, Reth, Timmer, & Schamel, under
revision
).
Acknowledgments
We thank Jose Antonio P´rez Gracia from the “servicio de dise˜o gr´fico y fotograf´a” of the
CBMSO for providing the photographs used in the figures. We thank Milagros Guerra and
Lara Rodenstein from the “servicio de microscop´a electronica” of the CBMSO for excellent
technical assistance. This study was supported in part by the Excellence Initiative of the Ger-
man Research Foundation (GSC-4, Spemann Graduate School) and EXC294 (BIOSS) to GJF
and WWS; the EU through grant FP7/2007-2013 (SYBILLA) to GJF, and WWS; and grant
BFU2009-08009 from the “Ministerio de Ciencia e Innovaci
´
n” to HMvS.
References
