1. Under liquid nitrogen, place freezing planchettes containing
samples in cryotubes containing frozen cryosubstitution
medium (0.2% glutaraldehyde plus 0.2% uranyl acetate in anhydrous acetone).
2. Transfer cryotubes to the Leica AFS and leave them at À90
C
for 3 days.
3. Raise the temperature of the chamber 5
C/ h up to À60
C.
4. Discard cryosubstitution medium and rinse samples and freezing planchettes with precooled anhydrous acetone at least three
times.
5. Remove freezing planchettes with precooled tweezers. Samples
should be free in the acetone by now, completely detached
from the freezing planchettes.
6. Add increasingly higher concentrations of HM20 in anhydrous
acetone to the cryotubes. Three steps are recommended: 30%,
60%, and 100% HM20 resin (at least 2–3 h for each concentration). All the resin solutions should be precooled to À60
C
before being added to the cryotubes containing the samples.
7. Add fresh, precooled 100% HM20 resin mix 4–5 times over the
next 24 h.
8. With a precooled glass Pasteur pipette remove samples from
cryotubes and place them in Coverwell imaging chambers with
a glass coverslip on top. Be sure to completely fill the chamber
with resin; no air bubbles should be seen after placing the glass
coverslip.
9. Attach the UV light module and adjust the temperature to
À50
C (temperature ramp 5
C/ h).
10. Polymerize at À50
C for 24 h followed by a slow warming up
to 18
C over the following 24 h. After 24–48 h under UV
light, the HM20 resin should be completely polymerized.
11. Remove resin blocks with samples from the imaging chambers.
12. Select samples in resin blocks using a dissecting microscope and
mount pieces of resin containing the samples on plastic mounting cylinders.
3.4 Post-Embedding
Sectioning and Heavy
Metal Staining for
Structural Analysis
1. Section high-pressure frozen/freeze-substituted samples
embedded in Eponate in an ultramicrotome (60–70 nm thick
section).
2. Collect sections on single slot specimen grids coated with
Formvar.
3. Stain sections by floating grids on a drop of 2% uranyl acetate in
70% methanol (see Note 8) for 5 min and rinse with 70%
methanol (30 s) and distilled water (10 s).
344
Marisa S. Otegui
samples in cryotubes containing frozen cryosubstitution
medium (0.2% glutaraldehyde plus 0.2% uranyl acetate in anhydrous acetone).
2. Transfer cryotubes to the Leica AFS and leave them at À90
C
for 3 days.
3. Raise the temperature of the chamber 5
C/ h up to À60
C.
4. Discard cryosubstitution medium and rinse samples and freezing planchettes with precooled anhydrous acetone at least three
times.
5. Remove freezing planchettes with precooled tweezers. Samples
should be free in the acetone by now, completely detached
from the freezing planchettes.
6. Add increasingly higher concentrations of HM20 in anhydrous
acetone to the cryotubes. Three steps are recommended: 30%,
60%, and 100% HM20 resin (at least 2–3 h for each concentration). All the resin solutions should be precooled to À60
C
before being added to the cryotubes containing the samples.
7. Add fresh, precooled 100% HM20 resin mix 4–5 times over the
next 24 h.
8. With a precooled glass Pasteur pipette remove samples from
cryotubes and place them in Coverwell imaging chambers with
a glass coverslip on top. Be sure to completely fill the chamber
with resin; no air bubbles should be seen after placing the glass
coverslip.
9. Attach the UV light module and adjust the temperature to
À50
C (temperature ramp 5
C/ h).
10. Polymerize at À50
C for 24 h followed by a slow warming up
to 18
C over the following 24 h. After 24–48 h under UV
light, the HM20 resin should be completely polymerized.
11. Remove resin blocks with samples from the imaging chambers.
12. Select samples in resin blocks using a dissecting microscope and
mount pieces of resin containing the samples on plastic mounting cylinders.
3.4 Post-Embedding
Sectioning and Heavy
Metal Staining for
Structural Analysis
1. Section high-pressure frozen/freeze-substituted samples
embedded in Eponate in an ultramicrotome (60–70 nm thick
section).
2. Collect sections on single slot specimen grids coated with
Formvar.
3. Stain sections by floating grids on a drop of 2% uranyl acetate in
70% methanol (see Note 8) for 5 min and rinse with 70%
methanol (30 s) and distilled water (10 s).
344
Marisa S. Otegui