Biomedical Engineering Reference
In-Depth Information
3. Wash cells with 0.2 M cacodylate buffer (pH 6.9) 3 × 5 min.
4. Treat samples with 0.3 % thiocarbohydrazide in 0.2 M cacodyl-
ate buffer (pH 6.9) for 20 min.
5. Wash cells with 0.2 M cacodylate buffer (pH 6.9) 3 × 5 min.
6. Treat cells with the reduced osmium for 1 h on ice in the dark-
ness (if one uses silver enhancement, the incubation time
should be 5 min).
7. Wash cells with 0.2 M cacodylate buffer (pH 6.9) 3 × 5 min.
8. Treat samples with 0.3 % thiocarbohydrazide in 0.2 M cacodyl-
ate buffer (pH 6.9) for 20 min.
9. Wash cells with 0.2 M cacodylate buffer (pH 6.9) 3 × 5 min.
10. Treat cells with the reduced osmium for 1 h on ice in the dark-
ness (if one uses silver enhancement, the incubation time
should be 5 min).
If you do not plan to use TEM or STEM tomography,
steps 6
-
10
could be omitted.
11. Rinse sample in 50 % ethanol 2 × 5 min.
12. Dehydrate cells stepwise with 70, 90, and 100 % ethanol
(3 × 5 min for each) for the dehydration of the specimens.
13. Prepare Epon solution as described in materials.
14. Prepare a 1:1 Epon/100 % ethanol mixture (acetone and
propylene oxide could dissolve plastic Petri dishes). Vortex
until thoroughly mixed and store all solutions in the fume
hood.
15. Treat sample with a 1:1 Epon/100 % ethanol mixture for 1 h.
16. Replace the 1:1 Epon/100 % ethanol mixture with Epon alone
for 4 h with one exchange of Epon after 2 h.
17. Keep the cells in Epon for 1-2 h at room temperature, and
then leave them in an oven at 60 °C for 12 h.
18. After 12 h of polymerization of the Epon, place a small droplet
of a fresh resin on the site where the examined cell is located,
and insert a resin cylinder (prepared before by polymerization
of the resin in a cylindrical mold) with a fl at lower surface;
leave the samples for an additional 18 h in the oven at
60 °C. The resin cylinder should be placed on the semi-
polymerized Epon just over the cell of interest. The central
position of the cylinder is important to ensure the possibility of
using the transmission light for the examination of Epon
samples. If the wall of the cylinder is situated near the cell of
interest, the light will be scattered.
19. Polymerize for 24 h at 60 °C.
