Biology Reference
In-Depth Information
6. Place the block holder back into the ultramicrotome trimming
block.
7. With the blade, trim the block tip to shape it as a trapeze
containing the cell-containing selected region (0.5 × 1.0 mm
approx.).
8. Remove the sample holder from the trimming block and load
it in the microtome arm. Place a new glass knife in the micro-
tome knife holder.
9. Trim the block surface by cutting 40 nm thick slices to polish
until the surface shines.
10. Cut 70 nm thick sections using a diamond knife or a glass knife
with an attached plastic boat filled with distilled water. Fill the
boat until the water surface shines like a mirror. This is impor-
tant because in these conditions the thin sections floating on
the water will reflect a particular color that indicates their
thickness. With the loop, pick a few silver or golden sections
and transfer them to a grid. If necessary, sections can be moved
around on the water surface to regroup them using a hair
glued to a wooden stick.
1. Centrifuge saturated uranyl acetate and lead citrate for 5 min
at 18,550 ×
g
.
2. Put a drop (25 μL) of uranyl acetate on a piece of Parafilm and
place the grid on the drop with the sections in direct contact
with the solution. Incubate for 25 min in a dish covered to
protect the uranyl acetate from light.
3. Blot the grid with Whatman filter paper to remove the uranyl
acetate (but do not allow it to dry completely).
4. Wash the grid quickly by consecutive immersion in four large
drops (300 μL each) of MilliQ water.
5. Dip the grid in a drop of lead citrate (200 to 100 μL) and
incubate for 90 s. Make sure that the grid is completely
immersed in the drop and cover the plate during the incuba-
tion to minimize precipitation of lead citrate due to contact
with oxygen.
6. Wash the grid quickly in four drops (300 μL) of MilliQ Water.
7. Blot the grid and place on a new piece of filter paper inside a
petri dish, with the sections facing up. Wait until it is dry before
imaging at the electron microscope (
see
Fig.
3a
).
3.6.3
Section Staining
