Biology Reference
In-Depth Information
6. Prepare 100 mL of Epon resin (
see
item 9
in Subheading
2.6.1
and
Note 19
).
7. Embedding: prepare a mixture of acetone-Epon (1:1).
Incorporate the mixture to cells and incubate overnight under
gentle rotation. Centrifuge and remove the acetone-Epon
mixture, then add only Epon (1 mL approx.). Incubate for 6 h
at room temperature with agitation. When incubation time is
over, centrifuge the samples, remove the resin, add fresh Epon,
and incubate overnight with rotation (
see
Note 20
).
8. Encapsulation: Centrifuge the samples and change Epon. Mix
by rotation for 2 h. Cut small (0.7 × 0.4 cm) paper labels and
write sample identification and date with a pencil. Put one
label inside each beem capsule. Centrifuge the samples and
remove approximately three quarters of the resin content.
Resuspend the cells in the remaining resin and transfer them to
the beem capsules. Pour enough cell-Epon mixture to fill the
pyramidal part of each capsule. Depending on the amount of
cells, 2 or 3 capsules can be filled per sample. Centrifuge to
pellet down the cells (to centrifuge, put closed capsules into
Eppendorf tubes) and completely fill the capsules with Epon
until a convex meniscus is formed. Make sure that the labels
are clearly visible.
9. Polymerization: incubate the samples at 60 °C for 48 h with-
out closing the beem capsules. Finally, the beem capsule is cut
open with a razor and removed to obtain a block ready for
ultramicrotomy (
see
Note 21
).
1. Load a resin block in the ultramicrotome holder and place in
the trimming block with the cell pellet facing upwards.
2. Trim away resin from the surface of the block tip using a single
edge razor blade, until you reach the cell pellet.
3. Prepare glass knives by cutting a glass stripe according to the
knife maker instruction manual.
4. Remove the sample holder from the trimming block and load
it in the microtome arm. Place a glass knife in the microtome
knife holder.
5. Trim the block surface by cutting 1 μm thick slices with the
glass knife. Using forceps or a pair of needles, take some sec-
tions and put in a drop of water on a microscope slide. Heat to
evaporate the water. Cover the section with a drop of toluidine
blue, heat on a warm plate for 30-120 s. Gently rinse with
distilled water to eliminate excess stain. Dry and observe in an
optical microscope. Select a region with many cells and locate
this area in the Epon block.
3.6.2
Ultramicrotomy
