Determination of Altered Mitochondria Ultrastructure by Electron Microscopy - Protein Misfolding and Cellular Stress in Disease and Aging - page 273

box in order to ensure stability. It is useful to attach a slip of
paper bearing a name or identification number into capsules
in order to differentiate between samples.
14. Use an exclusive polymerizer for polymerization. Always
maintain low humidity, confirmed by a thermostat, with the
use of a desiccant. Polymerized hard blocks should be pre-
served in a desiccator or in a storage bottle containing the
desiccant, as such blocks are softened by humidification. The
present author typically employs Method 1.
15. The angle between the superior and lateral aspects of the
pyramid should measure about 120°, and should be sharp
(i.e. lacking any roundness).
16. Glass knives outperform diamond knives for cutting semi-thin
sections. It is important that both the angles of elevation and
those of right and left sides remain at zero during the rough-
cutting process. Several versatile ultramicrotomes are available
(e.g. LKB Ultratomes I and III; Sorvall Porter-Blum MT-1,
MT-2, and MT-7; and Reichert OMU2 and OMU3).
17. When the surface becomes even, sections (approximately
1-mm-thick) can be cut. After a satisfactory glass knife has
been prepared, a trough is constructed to collect sections.
A piece (~3 × 0.7 cm) of black electrical adhesive tape (Scotch
Brand No. 3 or equivalent) is wrapped from one side to the
other of the cutting edge of the glass knife. When the tape is
wrapped correctly, its lower edge should be parallel to the
lower edge of the knife; the upper edge of the tape should
precisely reach the top of the cutting edge. To render the
trough waterproof, the region of the heel of the trough
should be sealed with paraffin or beeswax that has been melted
in a dish on a hot plate or in any paraffin oven. To ensure a
better seal, the paraffin or beeswax used for sealing the trough
must be heated until it fumes. These fumes are considered
carcinogenic; necessary precautions should be taken to avoid
inhaling them. Alternatively, without making a trough, cut
semi-thin sections and pick them up directly with sharp twee-
zers or a thin metallic rod, and place the sections onto a drop
of water on the slide glass. In this case, tissue sectioned at a
thickness of more than 1 mm (approximately 2.0-2.5 mm) is
easier to handle. The present author prefers using a thin
metallic rod without making a trough.
18. To create the toluidine blue staining solution, mix 100 ml dis-
tilled water with 0.5 g of sodium tetra-borate (borax) and then
dissolve 0.5 g of toluidine blue completely in this solution.
(a) Cut 1-2.5 mm-thick sections, place each section on the
surface of a drop of distilled water on a clean slide glass,
heat the sections on a heating unit, and fix the sections
until they are dry.