4. Remove the excess liquid by touching the grid with filter paper.
5. Place specimen grid onto a 10 μL drop of secondary antibody
conjugated to gold particles (5, 10, 15 nm in diameter) diluted
1:10 in 5% milk in PBT-T-0.1% for 1 h.
6. Rinse the grid with a continuous stream of PBS-T-0.5% for
1 min followed by rinsing with distilled water.
7. Post-stain grid with 2% uranyl acetate in 70% methanol and
Reynold’s lead citrate.
4 Notes
1. Cryotubes with cryosubstitution solutions can be stored in
liquid nitrogen for several months.
2. OsO 4 in acetone is highly volatile. Even when the acetonic
OsO 4 solution is kept in closed cryotubes, osmication of surrounding objects can easily happen. It is advisable to have a
liquid nitrogen dewar fully dedicated to the storage cryosubstitution tubes with OsO 4 to avoid contamination of other
reagents.
3. BDMA is recommended as the accelerator for the Eponate
12 resin mix instead of DMP-30 because BDMA has lower
viscosity and diffuses more rapidly into tissues.
4. Arabidopsis cotyledons and leaf tissues in general can be poorly
preserved due to the presence of large intercellular spaces filled
with air that collapse under the high pressure applied during
freezing. Either growing seedlings in liquid medium or infiltrating cotyledons or leaves with cryoprotectant using a syringe
without needle can greatly improve preservation quality during
high-pressure freezing.
5. High-pressure frozen material can be stored in liquid nitrogen
for months without suffering changes in cellular preservation.
6. Keeping the cryotubes in agitation greatly reduced the time to
complete the cryosubstitution successfully [21]. Agitation
modules are also being adapted to work within Leica AFS
units [22].
7. Unpolymerized resin waste, dirty gloves, and other resincontaminated items should be placed in the oven for 24 h for
polymerization before discarding them.
8. A methanolic solution of uranyl acetate is recommended
because it increases the contrast of membrane and cytoskeletal
elements more than aqueous solutions do.
346
Marisa S. Otegui
5. Place specimen grid onto a 10 μL drop of secondary antibody
conjugated to gold particles (5, 10, 15 nm in diameter) diluted
1:10 in 5% milk in PBT-T-0.1% for 1 h.
6. Rinse the grid with a continuous stream of PBS-T-0.5% for
1 min followed by rinsing with distilled water.
7. Post-stain grid with 2% uranyl acetate in 70% methanol and
Reynold’s lead citrate.
4 Notes
1. Cryotubes with cryosubstitution solutions can be stored in
liquid nitrogen for several months.
2. OsO 4 in acetone is highly volatile. Even when the acetonic
OsO 4 solution is kept in closed cryotubes, osmication of surrounding objects can easily happen. It is advisable to have a
liquid nitrogen dewar fully dedicated to the storage cryosubstitution tubes with OsO 4 to avoid contamination of other
reagents.
3. BDMA is recommended as the accelerator for the Eponate
12 resin mix instead of DMP-30 because BDMA has lower
viscosity and diffuses more rapidly into tissues.
4. Arabidopsis cotyledons and leaf tissues in general can be poorly
preserved due to the presence of large intercellular spaces filled
with air that collapse under the high pressure applied during
freezing. Either growing seedlings in liquid medium or infiltrating cotyledons or leaves with cryoprotectant using a syringe
without needle can greatly improve preservation quality during
high-pressure freezing.
5. High-pressure frozen material can be stored in liquid nitrogen
for months without suffering changes in cellular preservation.
6. Keeping the cryotubes in agitation greatly reduced the time to
complete the cryosubstitution successfully [21]. Agitation
modules are also being adapted to work within Leica AFS
units [22].
7. Unpolymerized resin waste, dirty gloves, and other resincontaminated items should be placed in the oven for 24 h for
polymerization before discarding them.
8. A methanolic solution of uranyl acetate is recommended
because it increases the contrast of membrane and cytoskeletal
elements more than aqueous solutions do.
346
Marisa S. Otegui