Biomedical Engineering Reference
In-Depth Information
small membrane-enclosed compartments, because the electron
opaque products of the peroxidase reaction tend to diffuse
from the place of antibody binding [
1
].
An advantage of HRP-DAB immunostaining is that when
the antibody is directed against an intraluminal epitope, the
stain delineates the profi le of the labeled structure. This facili-
tates the recognition of the organelle of interest in consecu-
tive serial sections and hence its 3D reconstruction. A
limitation of this method, however, is that the pre-embedding
technique requires the balance between the need to preserve
the intracellular structures (which ideally requires strong fi xa-
tion) and the necessity of preserving epitopes and allowing the
penetration of the antibody (both properties are usually nega-
tively affected by strong fi xatives). This can be overcome by
appropriate fi xation protocols, but avoiding immune labeling
and using instead chimeras containing both GFP and HRP
can also overcome this problem perhaps more elegantly. Since
HRP maintains its activity even in strongly fi xed cells, fi xation
will no longer be a problem. Another limitation is linked to
the use of HRP itself; if the epitope is extra luminal, the HRP-
induced oxygen radicals leading to the precipitation of DAB
might diffuse from the site of production, thus reducing the
precision of labeling. This can be avoided by pre-embedding
immune labeling with a second antibody tagged not with
HRP, but with nanogold (
see
above), or with a fl uorochrome
suitable for inducing DAB photo oxidation, as this reaction is
much less prone to the problem of diffusion.
An alternative is to conjugate the monovalent Fab frag-
ments of a secondary antibody with both a fl uorochrome and
nanogold, since after a suitable (silver or gold) “enhance-
ment” procedure (Nanoprobes, NY, USA), the latter becomes
visible by EM. A fl uorescent ultrasmall immunogold probe,
FluoroNanogold, has been used for immunocytochemistry
on ultrathin cryosections. FluoroNanogold has the proper-
ties of both a fl uorescent-dye-conjugated antibody for FM and
Fig. 1
(continued) (
m
) Position of the acceptor slot grid on the holders. (
n
,
o
) The donor grid with serial sections
is moving toward the on acceptor grid. (
p
,
q
) If the dry acceptor grid is used, the dirt (
brown lines
on the accep-
tor grid) on the water surface could be placed on the serial sections. (
r
-
t
). To avoid dirt on the sections, it is
better to place a small droplet of glass-handled distilled water on the acceptor slot grid. Using stereomicro-
scope, put a small droplet of water on the supporting fi lm of the acceptor grid. (
u
) Elimination of excessive
water and orientation of the donor grid in parallel to the acceptor grid with the help of the very narrow fi lter
paper (to avoid too fast elimination of water between slot grids which could give incorrect positioning of grids;
the photo is shown in panel (
x
)). (
v
) Position of serial sections on the acceptor grid after drying and elimination
of the donor grid. (
w
) Detachment of the acceptor grid with serial sections from the sticky holders. (
y
)
Application of semithin serial sections instead of ultrathin sections for CLEM. Collection of semithin sections is
shown. (
z
) Re-embedding of serial semithin sections
