Biomedical Engineering Reference
In-Depth Information
6. Touch water with the donor grid in such a way that sections
should be inside the slot, and move the slot grid away. The
droplet of water and sections will be inside the slot.
7. To avoid dirt on the sections, it is better to place a small drop-
let of glass-handled distilled water on the acceptor slot grid.
Using stereomicroscope, put a small droplet of water on the
supporting fi lm of the acceptor grid.
8. Put the donor grid with serial sections on acceptor grid as it is
shown in Fig.
1
.
9. Using sharp and very narrow (to avoid too fast elimination of
water between slot grids which could give incorrect position-
ing of grids) fi lter paper, orient slot in parallel to each other,
and eliminate the excess of water from the space between slot
grids.
10. Dry grids during at least 20 min.
11. Carefully eliminate the donor grid.
12. Take the dried acceptor grid and check whether sections are in
correct position.
13. Use serial semithin sections if the cell of interest is too tall
(Fig.
1y, z
;
see
Note 6
).
EM examination, EM tomography, or analysis of samples
under FIBSEM should be done according to the instruction of the
manufacturer (
see
Notes 7
and
8
).
4
Notes
1. It is necessary to make photos at all steps of the sample prepa-
ration and be very careful during all steps of preparation. If
cells were transfected with two fusion proteins tagged with
fl uorescent proteins of different colors, i.e., green and red, it
is possible to photo cells immediately after fi xation and made
Z
-stacking and then bleach both fl uorescent proteins with the
laser beam of maximal intensity in order to use the light waves
for the next fl uorochrome which will be used for labeling after
fi xation. Double labeling of live cells together with quadruple
labeling with four distinct antibodies (using light with length
of wave of 454, 488, 536, and 633) and double labeling
(using silver enhancement) within the immune EM result in
eight different markers within the same organelle. This
approach is doable with culture cells and rather diffi cult with
tissue sections.
2. Usually, the immunogold protocol is suitable for the labeling
of the large majority of antigens, while the immunoperoxidase
protocol allows good labeling of only antigens residing within
