4. Ultrathin sectioning and preparation of arrays
4A. Required materials and equipment:
Silanized coverslips (24 Â 65 mm; Aratome, Menlo Park, USA), or High performance Schott Nexterion coverslips coated with Aminosilane (Schott, Jena,
Germany)
Carbon rods (e.g. Ted Pella, #93010)
Carbon evaporator (e.g. Cressington Carbon Coater 308R)
Ultra Jumbo Diamond Knife or Histo Jumbo Diamond Knife (Diatome): The
choice of knife depends on the size of the block to be cut. The Ultra Jumbo Knife
has a 3 mm diamond edge. For larger block faces, the Histo Jumbo knife comes
with 2 diamond size options—6 and 8 mm. Even though its cutting range is
specified as 0.2–5 lm, a new Histo Jumbo knife will cut ultrathin
sections (70 nm).
Cryotrim 45 Diamond Knife (Diatome)—for trimming the block face
Ultramicrotome
Weldwood Contact Cement
Xylene
Thin paint brush
Slide warmer (Set at *60 °C)
Tween-20 (Electron Microscopy Sciences 25564)
4B. Preparations:
Carbon coated coverslips: Coat the coverslips with carbon (medium gray
color). Follow the instructions for the particular carbon evaporator. Store in dust
free boxes.
Note: If the coat is too light, this can cause problems with charging of the sample in the
SEM. If the coat is too dark, the immunofluorescence signal will be significantly
attenuated. The coverslip will be very hydrophobic, however we don’t recommend glow
discharge to increase hydrophilic properties, because this decreases the adhesion of
sections to the coverslips.
Glue: Dilute contact cement with xylene (*1:3 until easily applicable with a
paintbrush).
Tween water: 0.005% Tween 20 in water. Dissolve 10 ll Tween 20 in 200 ml
distilled water.
4C. Procedures:
Prepare the block for ultrathin sectioning: Trim the block around the tissue using
a razor blade. A smaller blockface (2 mm or less width) with the width of the
trapezoid greater than its height works best. Cut semithin sections until you reach
the tissue. Trim the block again at this point, to ensure that the block face is not
too big and the leading and trailing edge of the block face are parallel. The
cryotrim 45 diamond knife can be used for precision trimming (Fig. 6.10a).
Using a paint brush, apply the diluted glue to the leading and trailing sides of the
block pyramid. Let dry (5 min) and apply again.
174
K. D. Micheva and K. D. Phend
4A. Required materials and equipment:
Silanized coverslips (24 Â 65 mm; Aratome, Menlo Park, USA), or High performance Schott Nexterion coverslips coated with Aminosilane (Schott, Jena,
Germany)
Carbon rods (e.g. Ted Pella, #93010)
Carbon evaporator (e.g. Cressington Carbon Coater 308R)
Ultra Jumbo Diamond Knife or Histo Jumbo Diamond Knife (Diatome): The
choice of knife depends on the size of the block to be cut. The Ultra Jumbo Knife
has a 3 mm diamond edge. For larger block faces, the Histo Jumbo knife comes
with 2 diamond size options—6 and 8 mm. Even though its cutting range is
specified as 0.2–5 lm, a new Histo Jumbo knife will cut ultrathin
sections (70 nm).
Cryotrim 45 Diamond Knife (Diatome)—for trimming the block face
Ultramicrotome
Weldwood Contact Cement
Xylene
Thin paint brush
Slide warmer (Set at *60 °C)
Tween-20 (Electron Microscopy Sciences 25564)
4B. Preparations:
Carbon coated coverslips: Coat the coverslips with carbon (medium gray
color). Follow the instructions for the particular carbon evaporator. Store in dust
free boxes.
Note: If the coat is too light, this can cause problems with charging of the sample in the
SEM. If the coat is too dark, the immunofluorescence signal will be significantly
attenuated. The coverslip will be very hydrophobic, however we don’t recommend glow
discharge to increase hydrophilic properties, because this decreases the adhesion of
sections to the coverslips.
Glue: Dilute contact cement with xylene (*1:3 until easily applicable with a
paintbrush).
Tween water: 0.005% Tween 20 in water. Dissolve 10 ll Tween 20 in 200 ml
distilled water.
4C. Procedures:
Prepare the block for ultrathin sectioning: Trim the block around the tissue using
a razor blade. A smaller blockface (2 mm or less width) with the width of the
trapezoid greater than its height works best. Cut semithin sections until you reach
the tissue. Trim the block again at this point, to ensure that the block face is not
too big and the leading and trailing edge of the block face are parallel. The
cryotrim 45 diamond knife can be used for precision trimming (Fig. 6.10a).
Using a paint brush, apply the diluted glue to the leading and trailing sides of the
block pyramid. Let dry (5 min) and apply again.
174
K. D. Micheva and K. D. Phend