Capsule-Based Processing and Handling
Rat kidney was perfusion fi xed, excised, immersed in Karnovsky’s fi xative [ 13 ], and then refrigerated until subse- quently it was fully processed. Pig heart and skin samples were rapidly excised aſt er euthanasia, immediately immersed in Karnovsky’s fi xative, and refrigerated until subsequently pro- cessed. Tissue specimens were then cut into 1 mm 3 pieces suit- able for TEM preparation and entrapped into mPrep/s cap- sules between the 300 μ m pores molded into the capsule bottom and the removable screen, which also has 300 μ m pores ( Figure 1a ). T e capsules were then attached to a 12-channel Gilson Neo P12X200N Pipetman ( Figure 1b ) via mPrep/f fi lter couplers, which prevent accidental drawing of reagents into the pipettor. To identify each specimen, adhe- sive labels were attached to each capsule. T e labels used in this study (provided with mPrep capsules) included both a computer-readable 2D (datamatrix) barcode and human- readable alphanumeric characters.
Tissue was processed by simultaneously aspirating 100 μ l of each reagent from polyethylene reservoirs (troughs) into all capsules. T e pipettor was held upright with the capsules resting in the reservoir for the designated reaction time using a lab stand. T e reagent sequence was:
1. Fresh Karnovsky’s fi xative for 1 hr (to ensure full fi xation aſt er tissue cutting)
2. 3 × 5 min washes in Sorensen’s sodium phosphate buff er
3. 1% OsO 4 (wt/vol) in Sorensen’s sodium phosphate buff er for 1 hr
4. 3 × 10 min washes in deionized water 5. 1% (wt/vol) aqueous uranyl acetate for 1 hr 6. 3 × 10 min washes in deionized water 7. 1 each 15 min washes of 20%, 50%, 70%, and 90% acetone in deionized water
8. 3 × 20 min washes in 100% acetone 9. 1:2 Epon/Spurr’s [ 14 ] : acetone infi ltration for 1 hr
10. 1:1 Epon/Spurr’s : acetone infi ltration for 1 hr 11. 2:1 Epon/Spurr’s : acetone infi ltration for 1 hr 12. 100% Epon/Spurr’s resin infi ltration for 12 hrs at room temperature
13. 100% Epon/Spurr’s resin
Figure 1 : Tissue processing using mPrep/s capsules: (a) Empty mPrep/s capsule. The arrow indicates the location for entrapping specimens between the capsule bottom with 300 μ m pores, and the removable, adjustable 300 μ m pore screen. For clarity no specimen is shown. The tool to insert and remove the screen is not shown. (b) Twelve labeled mPrep/s capsules [s] attached to a 12-channel Pipetman held above the reagent reservoir. Each mPrep/s capsule contains a ~1 mm 3 specimen and 100 μ l reagent (en bloc uranyl acetate). Capsules are connected to the pipettor via an mPrep/f fi lter coupler [f]. (c) Twelve mPrep/s capsules fi lled with epoxy and inserted into the mPrep/bench just prior to ejection from the pipettor. (d) mPrep/s capsule clamped into a microtome chuck with the capsule trimmed away to expose the epoxy-embedded tissue. The arrow shows the faced epoxy block ready for sectioning.
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Aſt er the fi nal fi ll with resin, mPrep/s capsules were inserted into an mPrep/bench as shown in Figure 1c , and, without dis- pensing the resin, the fi lled capsules were ejected from the pipettor. T e mPrep/bench is a silicone 96-well plate that tightly fi ts the mPrep/s (and mPrep/g) capsule bottoms to keep fl uid retained in the capsules. T e mPrep/f couplers were then removed, and additional resin was added using a transfer pipette to fi ll the mPrep/s capsules to the top. T e mPrep/bench with resin-fi lled capsules was then transferred to a 60°C oven for ~12 hr in-capsule polymerization. T e Dieff enbachia plant tissue was prepared similarly except that 2% aqueous OsO 4 was used for secondary fi xation, and the resin was Epon 812 (50% Epon 812, 20% DDSA, 20%NMA/ MNA, 10% DBP with 2.5% DMP-10 accelerator). Sectioning . T e mPrep/s capsules with polymerized epoxy blocks inside were directly clamped in the microtome chuck of a Leica U7 ultramicrotome. T e block was trimmed and faced for microtomy in the standard way aſt er fi rst trimming away the end of the mPrep/s capsule ( Figure 1d ). T ick and thin sections were then cut using a diamond knife. For light microscopy, 0.5 μ m kidney sections were cut and collected on glass slides, stained with poly- chrome I (methylene blue, azure II, 10% methanol, 10% glycerol), and examined with an Olympus BH2 microscope equipped with a Nikon 700 DSLR camera. For TEM, 70 nm sections were collected on 200-mesh Cu grids. One or two grids were then inserted and stored in labeled mPrep/g capsules ( Figure 2a ), and the capsules in turn were stored in an mPrep capsule grid box (not shown) until staining.
Grid staining and TEM exami-
nation . Grid-containing mPrep/g capsules were connected via mPrep/f couplers to a single-channel pipettor ( Figure 2b ) or to an 8-channel Gilson Neo P8X200N Pipetman ( Figure 2c ).
www.microscopy-today.com • 2015 September
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