Capsule-Based Processing and Handling
Figure 2 : TEM grid staining using mPrep/g capsules: (a) mPrep/g capsule with two copper grids inserted. Forceps tips are visible to right. (b) mPrep/g capsule with grids fi lled with uranyl acetate stain [g] attached to a single-channel pipettor via an mPrep/f coupler [f]. (c) Eight labeled mPrep/g capsules containing a total of 16 grids attached to an 8-channel Pipetman via mPrep/f couplers. Capsules are above a reagent reservoir fi lled with uranyl acetate stain.
With the 8-channel pipettor, 16 grids were simultaneously stained by aspirating 35 μ l of reagent into each mPrep/g capsule from a reagent reservoir ( Figure 2c ), following this protocol:
1. 2.5% (wt/vol) uranyl acetate in 50% ethanol for 14 min in the dark
Table 1 : Process and reagent effi ciency to fi x and embed 12 specimens using mPrep/s and vial processing.
Manual Operations
Place specimens into mPrep/s or processing vials
Karnovsky’s fix 1× Sorenson’s buffer 3×
Osmium tetroxide buffer solution 1×
Deionized water 3× Uranyl acetate 1× Deionized water 3×
Graded acetones in water 4×
100% acetone 3× Graded epoxies 3× 100% epoxy 2×
Transfer specimens into embedding molds
Additional resin into mold and insert label *
Total User Operations and Reagent Consumed
Reagent Consumed (ml) Process or Operation mPrep Vial mPrep Vial
12 1 3
1 3 1 3
4 3 3 2
1 12 49
12 12 36
12 36 12 36
48 36 36 24
12 12 324 –
1.2 3.6
1.2 3.6 1.2 3.6
4.8 3.6 3.6 2.4
0 1.2 30.0 –
12.0 36.0
12.0 36.0 12.0 36.0
48.0 36.0 36.0 24.0
12.0 12.0 312.0
* mPrep includes optional “topping off” of capsules with additional resin and optional insertion of a label into resin prior to curing. Vial assumes 2 ml resin per embedding in fl at or BEEM-type molds.
2015 September •
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2. Deionized water rinses: 24 rapid aspiration and dispense rinses (~2 seconds/cycle) were done with fresh water, with the water replaced aſt er every 8 rinses.
3. Reynold’s lead citrate [ 11 ] for 9 min. Note that because grids were inside mPrep capsules, no hydroxide (NaOH) pellets were used, as commonly practiced with droplet staining to reduce carbon dioxide exposure that can lead to lead precipitates [ 12 ].
4. Rinses with deionized water as above 5. T e mPrep/g capsules were removed from the pipettor aſt er dispensing the fi nal rinse water.
6. Most grids were air dried in the mPrep/g capsule. When faster drying was required for immediate TEM imaging, extraneous water was wicked from grids by inserting a wedge of fi lter paper into the capsule and touching the paper where the grids contact the inside of the capsule.
Grids were examined at 80 keV in a Philips CM120 TEM and an FEI Tecnai 12 for rat and pig tissues. A JEOL 1200EX
Table 2 : Process effi ciency to stain 16 grids using mPrep/g processing and droplet staining.
Operation
Place grids in capsule or grid box
Uranyl acetate stain Water rinses
NaOH pellets setup Lead citrate stain Water rinses
Wick off excess water Store grids in box Total User Operations
mPrep/g capsule operations
16 1 1
Not necessary, sealed capsule
1 1
16 * Already in
storage capsule 20 or 36
16 113
* Wicking off excess water is optional with mPrep/g processing, hence the range in total user operations.
33 Droplet operations
16 16 16
1
16 16 16
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