reSolution_LNT_No1_en - Leica Microsystems

BIOLOGY
Fig. 2
Fig. 1 & 2: Distribution and colocalization of
GABA (B1) and Kir3.2 in dendrites of hippocampal
cells as revealed by the SDS-digested freezefracture
replica labeling technique. A, Immunoparticles
for the GABA (B1) subunit were found
in clusters (arrows) over the surface of dendritic
shaft (Den) and spine (s) of a putative pyramidal
cell. B, C, Double and triple immunogold labeling
for Kir3.2 (5 nm particles; double arrows), GABA
(B1) (10 nm; arrows), and PSD-95 (15 nm in C)
revealed that the two proteins co-clustered in
dendritic spines of pyramidal cells (B and C) and
associated to the site of glutamatergic synapses
indicated by immunoreactivity for PSD-95 (C).
Scale bars, 200 nm
4 reSOLUTION
in order to facilitate proper penetration.
Silver intensifi cation of the gold
particles is subsequently carried out
to produce a detectable particle size.
This method produces non-diffusible
labels, thus the precise site of the reaction
and the location of the protein
at extra- and perisynaptic sites can be
determined. Synaptic proteins, however,
cannot be detected using this
method, most likely due to the inaccessibility
of the epitopes in the synaptic
specializations of fi xed tissues 2 .
(ii) The postembedding immunogold
method overcomes the problems of
pre-embedding technique by reacting
immunochemicals with the antigens
exposed on the surface of the ultrathin
sections and then detecting synaptic
proteins with the same sensititvity as
that for non-synaptic molecules. This
also improves quantitative evaluation
of the protein densities.
However, in resin-embedded sections
substantial proportions of proteins are
buried and therefore not accessible
for antibodies, limiting the detection
sensitivity of this technique 2 . (iii) In
SDS-FRL, the brain tissue is frozen
with a high-pressure freezing machine (Leica EM HPM100)
then frozen samples are freeze-fractured in a replica machine
(Leica EM BAF060). Proteins are allocated to either
the protoplasmic faces (P-faces) or the exoplasmic faces
(E-faces) of plasma membranes. Molecules are immobilized
with a thin layer of carbon (3-5 nm) followed by a
further coating with a 2-nm-thick platinum/carbon layer for
shadowing the membrane faces and then this material is
strengthened with a 15 – 20 nm thick carbon deposit 3 . The
SDS-FRL technique has two major advantages compared to
conventional immunogold methods.
First, the sensitivity of the SDS-FRL is considerably higher
than that of the pre- and postembedding techniques, because
membrane proteins are exposed on the two-dimensional
surface of the replica (Fig. 1), making them readily
accessible to immunoreagents. In addition, epitopes are
denaturated by SDS, allowing antibodies known to be
suitable for immunoblot analysis to react similarly with
proteins immobilized on the replica membrane. Second,
synaptic and extrasynaptic proteins can simultaneously
be visualized and quantifi cation of immunogold density in
membrane segments can be achieved.
This immunocytochemical method, similarly to others, has
limitations. First, the identifi cation of labeled morphological
structures is diffi cult, therefore, it is necessary to use
marker proteins to facilitate the identifi cation of fractured
membranes 6 . Second, the separation of membrane proteins
to P-face or E-face is unpredictable: some proteins are preferentially
allocated to either the P-face, such as GABA (B1),
Kir3.2 4 or the E-face, such as AMPA receptors 5 , whereas
others, like gluRδ2 6 are localized to both faces. Thus, for
quantitative studies, the allocation of the molecules should
carefully be examined 7 .
Taken together, these three immunocytochemical techniques
provide complementary information about the cellular
and subcellular distribution of proteins and are widely
used for high-resolution qualitative and quantitative analysis
of receptor and ion channel localization and colocalization
in post- and presynaptic compartments of neurons.
Contact
Daniel Althof, Akos Kulik
Department of Anatomy and Cell Biology,
Institute of Neuroanatomy, Spemann Graduate School of
Biology and Medicine, University of Freiburg, Germany
Department of Physiology II, University of Freiburg,
Germany
akos.kulik@physiologie.uni-freiburg.de