Abstract

This study’s droplet method used a pipet tip box covered with Parafilm that was slightly dimpled to provide a depression to hold stain droplets. Grids were placed section-side down on droplets of 2% aqueous Uranyl Acetate (UA), for 10 minutes, picked up with forceps, and rinsed with a water stream from a transfer pipette. They were then placed on droplets of Reynolds Lead Citrate (LC) for 10 minutes and covered. The grids were then rinsed with .002 Normal NaOH, followed by a water stream rinse, blotted with filter paper, and stored until TEM examination. Two problems that come up using this method at times is damage to the sections as you pick up grid between staining and rinsing and having to add more rinses to the sections. With the mPrep/g method, 1 or 2 grids are placed directly into mPrep/g capsules at the microtome, into tapered grid slots that securely hold them even if a capsule is dropped. For staining, the capsules attach like pipette tips to lab pipettors. This study used a 200 μl 8-channel Gilson Pipetman to enable staining of 16 grids in 8 mPrep/g capsules. Grids were stained from reagents held in a 96-well plate (Fig 1). The first row of the 96-well plate held 40 μl of UA, the next 3 rows held 400 μl of water, then 40 μl of LC, then 3 more water rows with 400 μl of water. 8 mPrep/g capsules holding 2 grids each were attached to the 8-channel Pipetman, and dipped into the bottom of the UA reagent row. 35 μl of UA was then drawn into the capsules and held for 10 minutes. The UA was then expelled back into this row, and the capsules/pipette were moved to the first water row. In this row, 40 μl of water was rapidly drawn up and down 30 times (taking about 60 seconds) to rinse and agitate, and then repeated in the next two water rows. Then LC stain was drawn in and held for 10 minutes, followed by expelling and another 2x15 water rinses. The capsules were then rested on filter paper to drain off water, and removed and stored in the capsule storage box (which they came in) until TEM imaging.

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