good membrane contrast [49], followed by a double osmium step with thiocarbohydrazide as a linker, allowing the deposition of the second osmium [50]. Lastly, an
en bloc aqueous uranyl acetate step is performed followed by an en bloc lead
aspartate [51] at 60 °C.
The NCMIR processing protocol is as follows:
1. Primary fixation in aldehydes and washing in buffer
2. 2% osmium tetroxide 1.5% potassium ferrocyanide 1 h
3. Water washes, 5 Â 3 min
4. 1% thiocarbohydrazide 20 min at room temperature
5. Water washes, 5 Â 3 min
6. 2% osmium tetroxide 30 min at room temperature
7. Water washes, 5 Â 3 min
8. 1% uranyl acetate (aqueous) overnight at 4 °C
9. Water washes, 5 Â 3 min
10. Lead aspartate at 60 °C 30 min at room temperature
11. Water washes, 5 Â 3 min
12. Dehydrate through graded acetone series
13. Infiltrate with Durcupan resin
14. Polymerise 48 h in 60° oven
The washes in this protocol must be thorough to ensure full removal of the
solutions and the use of high quality water and agitation is essential. Glass containers also prevent precipitation onto the wall of the container. Fresh chemicals
ensure the best staining so the purchase of new chemicals on a regular basis can be
valuable. The working solutions should be prepared fresh just before use though
some solutions (potassium ferrocyanide and aspartic acid) can be stored as stocks at
4 °C for up to a month. While this protocol uses potassium ferrocyanide, potassium
ferricyanide has also been shown to work successfully [9].
Samples produced by this and other similar protocols become extremely brittle
and so must be handled with great care or they will easily shatter into pieces. In
addition, cracks are often seen in these samples [52, 53]. The staining achieved by
these protocols can be inconsistent due to problems with the infiltration of the
reagents. To give consistent staining throughout the sample Hua et al. [52] have
modified the protocol so that they separate the osmium and the ferrocyanide, putting the sample first in osmium alone and then without washing, placing it into the
ferrocyanide. They have also shown that a two-step uranyl acetate protocol helps.
They allow the uranyl acetate to penetrate overnight at 4 °C then heat the solution
to 50 °C.
Embedding into small moulds is useful, since after polymerization the sample
will have to be cut away from all the surrounding resin. Silicone Isolator Sheets
from Electron Microscopy Supplies (Cat # 70338) make a good mould when a
small hole is punched in them. These are 0.5 mm in thickness and can be stuck to a
glass slide (Fig. 5.2b).
5 Volume Scanning Electron Microscopy: Serial Block-Face …
127
en bloc aqueous uranyl acetate step is performed followed by an en bloc lead
aspartate [51] at 60 °C.
The NCMIR processing protocol is as follows:
1. Primary fixation in aldehydes and washing in buffer
2. 2% osmium tetroxide 1.5% potassium ferrocyanide 1 h
3. Water washes, 5 Â 3 min
4. 1% thiocarbohydrazide 20 min at room temperature
5. Water washes, 5 Â 3 min
6. 2% osmium tetroxide 30 min at room temperature
7. Water washes, 5 Â 3 min
8. 1% uranyl acetate (aqueous) overnight at 4 °C
9. Water washes, 5 Â 3 min
10. Lead aspartate at 60 °C 30 min at room temperature
11. Water washes, 5 Â 3 min
12. Dehydrate through graded acetone series
13. Infiltrate with Durcupan resin
14. Polymerise 48 h in 60° oven
The washes in this protocol must be thorough to ensure full removal of the
solutions and the use of high quality water and agitation is essential. Glass containers also prevent precipitation onto the wall of the container. Fresh chemicals
ensure the best staining so the purchase of new chemicals on a regular basis can be
valuable. The working solutions should be prepared fresh just before use though
some solutions (potassium ferrocyanide and aspartic acid) can be stored as stocks at
4 °C for up to a month. While this protocol uses potassium ferrocyanide, potassium
ferricyanide has also been shown to work successfully [9].
Samples produced by this and other similar protocols become extremely brittle
and so must be handled with great care or they will easily shatter into pieces. In
addition, cracks are often seen in these samples [52, 53]. The staining achieved by
these protocols can be inconsistent due to problems with the infiltration of the
reagents. To give consistent staining throughout the sample Hua et al. [52] have
modified the protocol so that they separate the osmium and the ferrocyanide, putting the sample first in osmium alone and then without washing, placing it into the
ferrocyanide. They have also shown that a two-step uranyl acetate protocol helps.
They allow the uranyl acetate to penetrate overnight at 4 °C then heat the solution
to 50 °C.
Embedding into small moulds is useful, since after polymerization the sample
will have to be cut away from all the surrounding resin. Silicone Isolator Sheets
from Electron Microscopy Supplies (Cat # 70338) make a good mould when a
small hole is punched in them. These are 0.5 mm in thickness and can be stuck to a
glass slide (Fig. 5.2b).
5 Volume Scanning Electron Microscopy: Serial Block-Face …
127