Microscopy 101
Figure 2: A depiction of the steps in conventional negative staining procedures.
Figure 1: HDL particle assembly. Lipid-free Apo A1 freely circulating in plasma mediates the assembly of nascent discoidal HDL particles through interac- tions with ABCA1 at the plasma membrane of cells, leading to extraction of phospholipid and cholesterol from the plasma membrane. The extracted phos- pholipid and cholesterol form a bilayer with polar heads facing the aqueous environment, and Apo A1 wraps around the hydrophobic tails to shield them from the aqueous environment. A plasma enzyme known as LCAT, interacting with ApoA, then facilitates the addition of a fatty acid to the free cholesterol in the phospholipid layer. The resultant cholesteryl ester (CE) migrates from the phospholipid bilayer to the hydrophobic space between the bilayer. As more cholesterol accumulates in the hydrophobic core, the particle adds more phos- pholipid and becomes spherical to accommodate the increasing volume of the CE in the core of the particle. Eventually, a mature spherical particle is created.
stain technique. To be able to rapidly perform the steps in the staining procedure, an organized work area for staining was prepared in advance (Figure 3). Te stain station was made
from two styrofoam box lids with a pipette box rack covered with parafilm placed inside the stain station. Te parafilm was pressed down into the holes of the rack to create recessed areas for placing drops of water and stain during the procedure. Two different sizes of round filter paper (Whatman 90 mm grade 1 qualitative filter paper and VWR 18.5 cm grade 410 qualitative filter paper) were torn into wedges for blotting sample, water, and stain from the TEM grids during the staining procedure. Te smaller wedges (Whatman 90 mm) were used for blotting excess sample and staining fluid from the grid and for stor- ing the grid. Te larger wedges (VWR 18.5 cm) were used for quickly blotting excess fluid from the water washes and the first drop of stain. Plastic petri dishes (10 cm) lined with the What- man 90 mm filter paper were assembled for storing the stained grids at the conclusion of the staining procedure. A 15 ml conical tube was filled with ddH2
O for use in the water washes
during the staining procedure. Sample concentration varied according to the type of material being stained, but for HDL particles, we usually prepared a solution of between 0.01 and 0.1 mg protein per ml of diluent, typically standard Tris buffer. Stain procedure. Formvar/carbon-coated 200-mesh cop-
per TEM grids (Electron Microscopy China #BZ11022a) were placed onto a parafilm-covered glass slide and set inside a 100 mm glass petri dish. To make the grids more hydrophilic, they were glow discharged in a glow discharge unit (Electron Microscopy Sciences EMS100) at 25 mA current for 2 minutes at negative polarity. Immediately aſter glow discharging the grids, the following steps were performed as rapidly as possible:
1. A glow discharged grid was floated carbon-side-down on top of a 15–20 µl drop of sample for 10–30 seconds.
2. Excess sample was blotted away by touching the edge of the grid with a small wedge (Whatman 90 mm) of filter paper (Figure 4A).
3. Te grid was quickly touched to the surface of the first drop of ddH2
Figure 3: Layout of the staining station and the materials needed for our modi- fied negative staining procedure using uranyl formate.
2020 September •
www.microscopy-today.com O (carbon-side-down) while still being held by the
forceps, and then one edge of the grid was rapidly touched to a large wedge of filter paper (VWR 18.5 cm) to wick away excess ddH2
O. Tis large wedge of filter paper was held per-
pendicular to the pipette tip box rack directly behind the 55
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