Hit “Start” and then immediately “Pause”. Machine will go to T1 setting and
hold. The instrument is subject to defective temperature monitoring;
accordingly, when it displays the T1 temperature, the temperature should be
independently verified using an independent probe or solutions of known
freezing point. If necessary, adjust settings to achieve desired temperature.
When all samples are in the machine, hit “Pause” again and program will start.
Troubleshoot—things freezing when they shouldn’t—if chamber temperature is verified, check that TF control knob isn’t set too high (too far right).
Set up a freezing box (foil-lined Styrofoam box with about 1″ 100%
ethanol and dry ice bath), load capsules into labeled holders, fill each
holder with about 5 ml absolute methanol and place in freezing bath to
cool (cover when not transferring samples).
Under a dissecting microscope, place a section with a modest amount of
cold 30% glycerol in a petri dish or on dental wax. Use a scalpel blade to
excise desired areas of a size suitable for ultramicrotomy from cryoprotected sections (remainder of section can be returned to vial and stored for
future use). Use a fine brush to transfer each sample to the tip of an
ACLAR spear (2 cm long  2 mm wide tapered at one end), blot the
brush and use it to wick off excess glycerol. Then transfer the loaded spear
to the first capsule (refer to capsule numbering scheme in manual, #1 is
marked by dots) and drop it in. As soon as one holder is loaded (8 samples), transfer it to the AFS chamber. Continue until all holders are loaded
and transferred to AFS. Hit “Pause” to start the programmed run.
** If using 100 µm sections for flat embedding, mount the cryoprotected
sections on 2 cm  6–8 mm ACLAR strips (both sides if necessary), and
insert upright into mesh-bottomed baskets in cans. Sections must be small
enough to be immersed while upright. Baskets (4/can) can be notched to
identify and cans tape-labeled. Sections can be wafer-embedded after
substitution and infiltration and either cured in the AFS or removed to a
chilled, external UV equipped chamber.
Once the program is started:
Chill any new solution in a separate can in the AFS for 5–10 min; also
provide a can to receive waste solutions.
Briefly chill 2 disposable pipets (1 for removal of solutions, 1 to transfer
new solutions).
2 Â 10 min changes of anhydrous methanol (3 changes if there are
moisture concerns).
After each change, use the new solution pipet to gently suck up and down
in the central tube in each capsule holder, to mix the solution.
Replace the final methanol rinse with uranyl acetate/methanol. After mixing in
each holder, close the machine, lower the glass plate, and detach it from the lifter.
Collect ethanol and methanol for hazardous waste disposal.
Clean used cans.
170
K. D. Micheva and K. D. Phend
hold. The instrument is subject to defective temperature monitoring;
accordingly, when it displays the T1 temperature, the temperature should be
independently verified using an independent probe or solutions of known
freezing point. If necessary, adjust settings to achieve desired temperature.
When all samples are in the machine, hit “Pause” again and program will start.
Troubleshoot—things freezing when they shouldn’t—if chamber temperature is verified, check that TF control knob isn’t set too high (too far right).
Set up a freezing box (foil-lined Styrofoam box with about 1″ 100%
ethanol and dry ice bath), load capsules into labeled holders, fill each
holder with about 5 ml absolute methanol and place in freezing bath to
cool (cover when not transferring samples).
Under a dissecting microscope, place a section with a modest amount of
cold 30% glycerol in a petri dish or on dental wax. Use a scalpel blade to
excise desired areas of a size suitable for ultramicrotomy from cryoprotected sections (remainder of section can be returned to vial and stored for
future use). Use a fine brush to transfer each sample to the tip of an
ACLAR spear (2 cm long  2 mm wide tapered at one end), blot the
brush and use it to wick off excess glycerol. Then transfer the loaded spear
to the first capsule (refer to capsule numbering scheme in manual, #1 is
marked by dots) and drop it in. As soon as one holder is loaded (8 samples), transfer it to the AFS chamber. Continue until all holders are loaded
and transferred to AFS. Hit “Pause” to start the programmed run.
** If using 100 µm sections for flat embedding, mount the cryoprotected
sections on 2 cm  6–8 mm ACLAR strips (both sides if necessary), and
insert upright into mesh-bottomed baskets in cans. Sections must be small
enough to be immersed while upright. Baskets (4/can) can be notched to
identify and cans tape-labeled. Sections can be wafer-embedded after
substitution and infiltration and either cured in the AFS or removed to a
chilled, external UV equipped chamber.
Once the program is started:
Chill any new solution in a separate can in the AFS for 5–10 min; also
provide a can to receive waste solutions.
Briefly chill 2 disposable pipets (1 for removal of solutions, 1 to transfer
new solutions).
2 Â 10 min changes of anhydrous methanol (3 changes if there are
moisture concerns).
After each change, use the new solution pipet to gently suck up and down
in the central tube in each capsule holder, to mix the solution.
Replace the final methanol rinse with uranyl acetate/methanol. After mixing in
each holder, close the machine, lower the glass plate, and detach it from the lifter.
Collect ethanol and methanol for hazardous waste disposal.
Clean used cans.
170
K. D. Micheva and K. D. Phend