Microscopy101
Improved Negative Stain Electron Microscopy Procedure for Detecting Surface Detail on High Density Lipoproteins
Rachel Hart Vanderbilt University Medical Center, 1161 21st
rachel.c.hart.1@vumc.org
Abstract: While conventional negative stain electron microscopy (NSEM) has been used extensively for imaging and characterizing high density lipoprotein (HDL) particles, traditional methods have not pro- vided high-level particle surface detail. To obtain greater detail of HDL particles, we developed a modified negative staining procedure using a uranyl formate staining solution that maintains particle morphology and provides improved surface detail. The additional surface detail has allowed us to perform single-particle analysis on whole HDL particle preparations. It has also allowed us to explore interactions between HDL particle surface proteins such as Apo A with proteins that interact with Apo A such as lecithin-cholesterol acyltransferase (LCAT).
Keywords: transmission electron microscopy (TEM), negative stain, high density lipoprotein (HDL), lecithin-cholesterol acyltransferase (LCAT), single-particle analysis
Introduction Negative stain electron microscopy (NSEM) has been used
extensively for characterizing lipoprotein particles [1,2], includ- ing high density lipoproteins (HDLs) [3]. HDLs are small six- to twelve-nanometer lipid-protein complexes that transport choles- terol and other lipids from peripheral tissue for removal, offer- ing a protective effect against atherosclerosis and cardiovascular disease. Structurally, HDLs are composed of a phospholipid shell, neutral lipid core, and at least two copies of apolipoprotein A (Apo A) on its surface. Along with Apo A, HDL particles display a wide variety of other surface proteins from a proteome of almost 100 different proteins [4]. Visualizing the location of these surface proteins and their interactions with extraneous proteins, such as ATP-binding cassette transporter A1 (ABCA1) and lecithin- cholesterol acyltransferase (LCAT), is important in uncovering the mechanisms by which HDL particles are assembled, mature, and promote cholesterol removal from cells (Figure 1). Negative staining is a method in which the background is
stained and the specimen is leſt unstained. Tus, in a negative stain image, the specimen appears light while the background appears dark. In conventional negative staining procedures, the sample is adhered to a substrate such as formvar on a transmis- sion electron microscope (TEM) grid, and an electron-dense staining solution is applied. Tis solution typically includes a heavy metal such as tungstate or uranyl acetate. Excess stain is wicked away, leaving a build-up of stain around the edges of the particle that provides contrast during TEM imaging (Figure 2). While NSEM provides contrast along the edges of particles, conventional NSEM methods have not yielded high-resolution details on the HDL particle surface. Over the years several
54 doi:10.1017/S1551929520001303
groups have modified traditional negative stain methods to attempt to obtain more detail. Ohi et al. highlighted the use of uranium-based stains, namely uranyl formate, and image clas- sification methods to provide higher-resolution images of small, heterogeneous proteins [5]. For imaging lipoproteins, Rames et al. developed an “optimized negative staining procedure” using uranyl formate as the negative stain. Tey altered sev- eral aspects of traditional negative staining methods to reduce the formation of HDL aggregates (rouleaux) and provide bet- ter particle detail [6]. Working from the Ohi and Rames stud- ies, we implemented a modified uranyl formate-based negative staining procedure for visualizing and analyzing particles. Tis method improves overall HDL imaging and provides greater surface detail without altering particle morphology.
Materials and Methods HDL isolation. Human EDTA plasma was subjected to a
one-step gradient density ultracentrifugation procedure. HDL fractions were collected from the bottom of the tube (density 1.063 to 1.210 g/ml). Te HDL fractions were concentrated by ultrafiltration and dialyzed into standard Tris buffer [7]. Phosphotungstic acid negative stain electron microscopy.
Before staining, dip formvar-carbon-coated copper electron microscopy grids in ethanol to make the grid surface hydro- philic. Float the grids carbon-side-down onto a 40 µl drop of HDL particles in buffer for 30 to 60 seconds. Remove excess fluid from the grids by touching the side of the grid to a wedge of filter paper. Float the grid with adsorbed particles carbon-side-down on a 40 µl droplet of 2% phosphotungstic acid stain at pH 6.0 for 60 seconds. Remove excess stain with a wedge of filter paper and allow the grids to dry before viewing with the TEM [7]. Preparing 0.7% uranyl formate stain. In low-light con- ditions, weigh 37.5 milligrams of uranyl
Microscopy Sciences cat. #22450) into a foil-covered beaker, and add 5 ml of boiled distilled deionized water (ddH2
formate (Electron O). Stir
for five minutes and add approximately five μl of 5 M sodium hydroxide to bring the pH of the solution to ∼4.5. A slight color change to a darker yellow will be observed. Stir for an addi- tional five minutes, and filter solution through a 0.22 µm sterile syringe filter attached to a 1 ml sterile syringe into a foil- covered conical tube. It is important to keep the stain covered in foil as it is light-sensitive and can precipitate if exposed to light. Preparing the work area for staining. Torough but rapid application of stain and wash fluid is critical to the negative
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