Capsule-Based Processing and Handling
Figure 5 : Vampire bat ( Desmondontidae ) brain prepared using mPrep/g grid staining.
and intensely. This is expected because the chemistry of sample preparation and staining were unchanged; only the devices used for delivering reagents and the reagent volumes were different. With mPrep capsule processing there is also the ability to improve quality and experimental reproduc- ibility. This is because mPrep capsule pipetting provides simultaneous reagent delivery to multiple specimens thus providing identical reagent reaction times for all specimens. Secondly, because reagent exchanges occur very rapidly (≤1 second), the potential for accidental air drying that can occur with vial processing is nearly eliminated. Thirdly, because specimens are safely entrapped, the potential for damage caused by the specimen sticking to the side of vials, floating on the reagent surface, or being accidently sucked into transfer pipettes, is eliminated. Similarly, because grid handling is nearly eliminated, the probability for grid dam- age or loss is reduced, while simultaneous staining improves reproducibility. Reagent effi ciency . Because mPrep/s capsule volume is sized for TEM specimens, specimen processing only required 30 ml for 12 specimens (2.5 ml per specimen), as compared to a calculated reagent consumption of 26 ml per specimen for vial processing based on using only 1 ml per each vial reagent exchange ( Table 1 ). T is 10-fold reduction is substantial, yet it could be further reduced because as little as 10 μ l per reagent exchange can be used with small specimens entrapped at the bottom of the capsules. T us, the potential to reduce consumption by about 100-fold exists, which is especially desirable with expensive reagents such as antibodies, gold labels, or toxic compounds. Operational effi ciency . Simultaneous processing with multi-channel pipetting not only can improve reproducibility, but also can greatly reduce labor operations ( Tables 1 and 2 ).
2015 September •
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Figure 6 : Dieffenbachia (dumb cane) prepared using mPrep/s and mPrep/g capsules.
Note that the simple counting of operations in Tables 1 – 2 does not fully characterize the improved operational ease. For mPrep reagent exchanges, minimal attention is required because specimens and grids are safely encapsulated, and only a simple depression of the pipettor aspirate-dispense button is required. With mPrep/s capsules, specimens are only directly handled when inserted into the capsule and when the block was prepared for sectioning. Sectioning preparation is also easier because mPrep/s capsules are directly chucked into the microtome and faced through the capsule ( Figure 1d ). With respect to grid processing, grids are only handled when they are inserted into an mPrep/g capsule and when removed from the microtome to insert into the TEM stage, thus greatly reducing operator attention in comparison to most staining methods. An additional effort reduction is that Prep/g capsules reduce air exposure so that lead citrate staining does not require hydroxide pellets to sequester carbon dioxide [ 12 ].
Enabling multiple protocol processing using microtiter
plates . In the present study, specimens and grids were processed using multi-channel pipettes with reagent drawn from a single reagent reservoir ( Figures 1 b and 2 c), thus all specimens and grids were processed with the same reagents. However, by using the innovation of the 96-well microtiter plate [ 7 – 9 ], it is just as easy to deliver diff erent reagents to each of the mPrep capsules. T is is simply achieved by placing diff erent reagents, titrations, or stains into individual microtiter wells, and by using an 8- or 12-channel pipettor to draw reagents into mPrep capsules from diff erent wells. T is is shown in the Application Notes section of the Microscopy Innovations website where a template can guide protocols such as immuno-labeling [ 15 ]. T is template methodology can be extended to many specimen or grid applications, such as the study where McClain simultaneously
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