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amplifi cation. Secondary antibodies with differently sized gold
particles can be used for double immunostaining. This is usually
done by using primary antibodies raised in different animals (e.g.,
in mouse and in goat). Instead of secondary antibodies, Protein
A-gold or Protein G-gold can be used. Both bind to a single site at
the Fc region of antibodies of various host species (but not to all!)
which means that signal amplifi cation is not possible. Streptavidin-
gold can be used in combination with a biotinylated antibody.
An alternative to conventional colloidal gold is fl uorescent
nanogold particles [
7
] and quantum dots [
8
]. These probes allow
studying the same specimen by fl uorescence microscopy and by
EM (visualization of quantum dots requires the use of an energy-
fi ltered transmission electron microscope).
1.5 Postembedding
Immunogold Labeling
The most frequently used method for EM cytochemistry is indirect
immunogold labeling of ultrathin sections of resin-embedded mate-
rial (Fig.
1b, c
). A prerequisite for successful immunolabeling is the
preservation of the antigen, especially on EM sections where only
few binding sites are available for recognition by the antibody. For
postembedding immunogold labeling, high-pressure frozen mate-
rial and cryosubstituted material are frequently used because ultra-
rapid freeze fi xation is considered to preserve structure and antigen
binding sites more effi ciently than chemical fi xation [
9
]. But often
a compromise between preservation of cytoarchitecture and preser-
vation of antigen has to be made. Before sectioning, cells or tissues
are embedded in polar acrylic resins [
1
,
4
,
5
,
9
-
12
]. Epoxy resins
because of their hydrophobicity and the high temperature necessary
for polymerization are usually less suited for immunogold labeling.
In the following we describe a protocol for immunogold label-
ing of resin-embedded sections. Further information can be found
in refs. [
1
-
16
] as well as in protocols provided on the homepages
of EM laboratories or antibody manufacturers. Protocols for pre-
embedding immunogold labeling are to be found elsewhere [
17
]
as well as immunolabeling of cryosections [
18
-
20
]. The latter
method offers the advantage that epitope antigens are much better
preserved and accessible for antibody binding but requires the use
of a cryostat microtome. Methods to quantify immunogold local-
ization are described in ref. [
21
].
2
Materials
All solutions should be prepared with double distilled water if
available (
see
Note 1
). Reagents should be of analytical grade and
can be stored at room temperature, except bovine serum albumin
(BSA, 4 °C) and antibodies (4 °C or frozen,
see
Subheading
2.3
).
Buffers can be prepared as 10× stock solutions and supplemented
by 0.02 % NaN
3
for longer storage. All other solutions should be
freshly prepared.
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