[20] followed by embedding in methacrylate-based low-temperature UV-curing resins, such as Lowicryl HM20, are preferred for
immunogold-labeling applications.
3.3.1 For Structural
Analysis Using Dry Ice and
Styrofoam Box
1. Place freezing planchettes with frozen samples in cryotubes
containing 1.5 mL of 2% OsO 4 in acetone. Be sure to keep
cryotubes in liquid nitrogen during planchette transferring and
to precool the tip of the tweezers in liquid nitrogen before
touching the freezing planchettes.
2. Place the cryovials in an aluminum block precooled in dry ice
(at À80
C) inside a styrofoam box and keep it there overnight.
3. Transfer aluminum block with cryotubes inside a small Styrofoam box containing a few pellets of dry ice and place it on a
rocking shaker under gentle agitation inside a fume hood for
6 h (see Note 6).
4. Once the aluminum block has reached room temperature,
discard substitution medium and rinse samples with fresh anhydrous acetone at least five times (every 5 min).
5. Remove freezing planchettes (freezing planchettes can be
reused if they are cleaned by sonication in methanol or
acetone).
6. Rinse samples one more time with fresh acetone.
7. Add increasing concentration of Eponate 12 resin mix (without
accelerator) in anhydrous acetone and keep the samples at least
4 h in each resin concentration: 10% Eponate 12 resin mix, 25%
Eponate 12 resin mix, 50% Eponate 12 resin mix, 75% Eponate
12 resin mix.
8. Add 100% Eponate 12 resin mix (without accelerator) for at
least 8 h.
9. Add 100% Eponate 12 resin mix with accelerator for at least
12 h (repeat this step twice).
10. Place samples in flat embedding molds and polymerize at 60
C
for 24 h (see Note 7).
11. Select samples in resin blocks using a dissecting microscope and
mount pieces of resin containing the samples on plastic mounting cylinders.
3.3.2 For
Immunolabeling Using the
Leica AFS
Cryosubstitution for immunolabeling can also be performed using
“custom” freeze-substitution devices as explained in Subheading
3.3.1. However, if a low-temperature UV-curing resin is used, it is
advisable to use an automatic freeze-substitution device such as the
Leica AFS. This device allows for a very precise control of the
temperature during freeze-substitution and resin embedding. In
addition, it includes UV light attachment that can be directly
positioned on top of the sample chamber for resin polymerization.
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
343
immunogold-labeling applications.
3.3.1 For Structural
Analysis Using Dry Ice and
Styrofoam Box
1. Place freezing planchettes with frozen samples in cryotubes
containing 1.5 mL of 2% OsO 4 in acetone. Be sure to keep
cryotubes in liquid nitrogen during planchette transferring and
to precool the tip of the tweezers in liquid nitrogen before
touching the freezing planchettes.
2. Place the cryovials in an aluminum block precooled in dry ice
(at À80
C) inside a styrofoam box and keep it there overnight.
3. Transfer aluminum block with cryotubes inside a small Styrofoam box containing a few pellets of dry ice and place it on a
rocking shaker under gentle agitation inside a fume hood for
6 h (see Note 6).
4. Once the aluminum block has reached room temperature,
discard substitution medium and rinse samples with fresh anhydrous acetone at least five times (every 5 min).
5. Remove freezing planchettes (freezing planchettes can be
reused if they are cleaned by sonication in methanol or
acetone).
6. Rinse samples one more time with fresh acetone.
7. Add increasing concentration of Eponate 12 resin mix (without
accelerator) in anhydrous acetone and keep the samples at least
4 h in each resin concentration: 10% Eponate 12 resin mix, 25%
Eponate 12 resin mix, 50% Eponate 12 resin mix, 75% Eponate
12 resin mix.
8. Add 100% Eponate 12 resin mix (without accelerator) for at
least 8 h.
9. Add 100% Eponate 12 resin mix with accelerator for at least
12 h (repeat this step twice).
10. Place samples in flat embedding molds and polymerize at 60
C
for 24 h (see Note 7).
11. Select samples in resin blocks using a dissecting microscope and
mount pieces of resin containing the samples on plastic mounting cylinders.
3.3.2 For
Immunolabeling Using the
Leica AFS
Cryosubstitution for immunolabeling can also be performed using
“custom” freeze-substitution devices as explained in Subheading
3.3.1. However, if a low-temperature UV-curing resin is used, it is
advisable to use an automatic freeze-substitution device such as the
Leica AFS. This device allows for a very precise control of the
temperature during freeze-substitution and resin embedding. In
addition, it includes UV light attachment that can be directly
positioned on top of the sample chamber for resin polymerization.
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
343