Biomedical Engineering Reference
In-Depth Information
20. Carefully pick up the resin from the Petri dish and glass; this is
easy to do by gentle bending of the resin cylinder to and fro.
The resin block and the empty MatTek Petri dish after block
detachment could be separated easily. If the cover glass with a
coordinated grid cannot be detached from the cells included
into the resin, the latter should be placed into commercially
available hydrofl uoric acid (do not use glassware for this) for
30-60 min.
21. Remove the glass bottom of tissue culture dishes by hydrofl uoric
acid for 30-60 min.
22. Control the completeness of the glass dissolution under a
stereomicroscope.
23. Wash the samples in water after the complete removal of the
glass.
24. Leave the samples in 0.2 M HEPES buffer (pH 7.3) for 60 min
to neutralize the hydrofl uoric acid.
25. Wash the samples in water and allow them to dry. The round
basement of the sample with diameter of about 1 cm or more
should not be cut.
1. Place the embedded sample under a stereomicroscope using a
fi ber optic illuminator.
2. If the gridded cover slip was used, the furrows on the cover
slips are fi lled with Epon, and after polymerization and dissolu-
tion of the glass or its detachment, the furrows appeared as
combs because Epon forms a replica from the gridded glass.
The relief of grid is visible under a stereomicroscope. Using
numbers and letters on the grid, it is easy to fi nd the position
on the surface of the sample where the cells are situated.
3. Use the composite of bright-fi eld DIC images or manual draw-
ings of the region of interest to locate the region of interest on
the Epon block. Another possibility for the identifi cation of
our cell could be the overall general pattern of the surrounding
cells and the localization of the cell within the cover slip. The
cell of interest could be surrounded by scrapping of the ring.
Finally, the position of the cell could be determined on the
basis of rectangular coordinates made by scrapping of the con-
fl uent monolayer of cell. After scrapping, the cell of interest is
easily identifi ed. Instead of a ring, one could make a perpen-
dicular coordinated grid (
see
Note 4
).
3.6 Identifi cation
of the Cell of Interest
on Epon Blocks
3.7 Sectioning
1. Place the sample under the transmission light microscope.
2. Find the cell of interest among the cells within the sample
according to the coordinated grid (Figs.
3a
and 4a) or pattern
of the cell layer (Fig.
3b, c
) or using scrapping (Fig.
3c-f
).
