Method for Combined Observation of Serial Sections of Stented Arteries Embedded in Resin by Light Microscopy and Transmission Electron Microscopy

Samples

Stents in the porcine coronary artery were used for the sample in this study. All procedures were approved by the Institutional Animal Care and Use Committee where the study was conducted.

Fixation

Porcine hearts with coronary stents were harvested (Figure 1A-1), perfused with saline, and then placed in an ice-cold 2% paraformaldehyde and 1.25% glutaraldehyde mixed solution for 60 min. After perfusion fixation, the stented arteries were removed from the heart and immersed in a freshly prepared ice-cold fixative overnight (Figure 1A-2). We postfixed the stented arteries in 1% osmium tetroxide for approximately 60 min on ice (Figure 1A-3). All of these fixatives were dissolved in 0.1 M phosphate buffer at a pH of 7.2–7.4.

OPEN IN VIEWER

Figure 1.

Schematic representation of the resupinate method and imaging of Quetol 651-embedded blocks of a stented artery postfixed with osmium. (A-1) Removing the stented artery from the heart. (A-2) Fixation with a paraformaldehyde and glutaraldehyde mixed solution. (A-3) Postfixation with osmium. The specimen turns black when immersed in an osmium solution used as postfixation. (A-4) Resin embedding and dividing. (A-5) Making serial sections. (A-6) Deplastization and immersion in periodic acid and hydrogen peroxide for removal of osmium. (A-7) Staining for preparation of LM. (A-8) Trimming the area of interest in sections with the resupinate method and staining preparation of TEM. (B) Photograph showing that the whole stented artery postfixed with osmium is black. LM = light microscopy; TEM = transmission electron microscopy.

Resin Embedding

After fixation, the stented arteries were dehydrated through a graded acetone series of 70%, 80%, 90%, and 2 × 100% for 60 min each. To make a resin mixture, 48.1 g of Quetol 651 (Nisshin EM, Tokyo, Japan), 55.4 g of methyl nadic anhydride (Nisshin EM), 13.5 g of nonenyl succinic anhydride (Heico Chemicals, PA), and 1.5 ml of dimethylaminomethyl phenol-30 (Nisshin EM) were gently stirred on a magnetic stirrer immediately before use. The stented arteries were immersed in acetone and Quetol 651 resin at the following ratios: (1) 3:1 (v/v) acetone: Quetol 651 resin for 20 min, (2) 1:1 (v/v) acetone: Quetol 651 resin for 20 min, and (3) 1:3 (v/v) acetone: Quetol 651 resin for 20 min.

The stented arteries were infiltrated with Quetol 651 resin for 60 min followed by immersion in fresh Quetol 651 resin overnight. The stented arteries were also embedded in Quetol 651 resin for 60 min and transferred into a polypropylene tube (PP-10; Maruemu, Osaka, Japan). This tube was filled with a freshly prepared Quetol 651 resin, placed in a desiccator to exclude air, and then capped. Polymerization was carried out at 60°C in an electric oven. Following polymerization, the stented arteries were cut by a diamond band saw (V-19; Luxo, Aichi, Japan; Figure 1A-4).

Sectioning

Blocks of stented arteries (Figure 1B) were serially sectioned at approximately 4-µm thickness using a rotary microtome (RM2255; Leica, Wetzlar, Germany), equipped with a tungsten carbide D profile blade. The serial sections were transferred onto adhesive-coated glass slides (MAS-coated glass slides; Matsunami Glass, Osaka, Japan) for LM and noncoated glass slides (regular glass slides; Matsunami Glass) for TEM (Figure 1A-5).

Staining and LM

The sections for LM were covered with polyethylene foil and gently flattened by pressing the fingers. Subsequently, these slides were pressed with a slide press (object holder press; Kulzer, Wehrheim, Germany) and dried in an electric oven at 60°C overnight. For deplasticization, the sections were immersed in 10% potassium hydroxide in ethanol for 15 min at 40°C. The sections were immersed in 100% ethanol for five times and then a graded ethanol series (90%, 80%, and 70%), followed by rinsing the sections in running water and distilled water. The deplasticized sections were placed in periodic acid for 30 min and hydrogen peroxide for 30 min for removal of osmium from the tissue (Figure 1A-6). The sections were stained with hematoxylin and eosin (HE) and Elastica–Masson trichrome (E-MT) according to a conventional protocol (Figure 1A-7).

TEM

According to a conventional method, Quetol 812 resin (Nisshin EM) was prepared immediately before use (Luft 1961). After sweeping with a brush to remove the pieces of stent, sections for TEM were re-embedded in Quetol 812 resin by the resupinate method (Di Sant’Agnese and De Mesy-Jensen 1984; Lehmbecker et al. 2014). Thin tissue sections were cut on an ultramicrotome using a diamond knife (Diatome; Hatfield, PA) and mounted on a grid. Thin sections were further stained with an equal mixture of 4% uranyl acetate and acetone for 30 s and lead citrate for 2 min. In the stained sections, morphology of endothelial cells and smooth muscle cells in the coronary artery was examined by TEM (HT7700; Hitachi High-Technologies, Tokyo, Japan; Figure 1A-8).

LM

After rinsing thin sections in periodic acid and hydrogen peroxide, the black color of osmium in tissue appeared to be removed. For HE staining, sections that were treated with removal of osmium yielded more detailed information compared with sections that were treated without removal of osmium (Figure 2A and B). For E-MT staining, the black color of osmium in tissue was also decreased after placing the sections in periodic acid and hydrogen peroxide (Figure 2C and D). This method enabled assessment of cells and the extracellular matrix, including flat-shaped endothelial cells, spindle-shaped smooth muscle cells, fibroblasts in the tunica adventitia, connective tissue, and myocardium (Figure 2B and D). HE and E-MT staining from the sections that were treated with removal of osmium resulted in an acceptable diagnostic quality.

OPEN IN VIEWER

Figure 2.

Comparison of light microscopy images of a stented artery with or without removal of osmium. (A, C) Images of a stented artery treated without removal of osmium. The black color of osmium remains in the media of the coronary artery and myocardium. (B, D) Images of a stented artery treated with removal of osmium. The black color of osmium cannot be seen. (A, B) hematoxylin and eosin staining. (C, D) Elastica–Masson trichrome staining. En = endothelium of tunica intima; Me = tunica media; Ad = tunica adventitia; My = myocardium; Bars = 100 μm.

TEM

The sections that were used for LM could be observed using TEM as clearly as the standard method. The nucleus and basement membrane of smooth muscle cells that were treated with osmium postfixation were clearly visible (Figure 3A). The inner cristae membrane in the mitochondria of smooth muscle cells in the tunica media was easily identified (Figure 3B). Myofilaments and cytoplasmic membranes in smooth muscle cells that were fixed with osmium were easily detected with negative contrast (Figure 3C). Additionally, fibroblasts and collagen fibers in the tunica adventitia were observed (Figure 3D).

OPEN IN VIEWER

Figure 3.

Transmission electron microscopy images from a specimen fixed with osmium postfixation. (A) The nuclear membrane (arrows) and basement membrane (arrowheads) of a smooth muscle cell in the tunica media can be seen. (B) The inner cristae membrane (arrow) in the mitochondria of a smooth muscle cell in the tunica media can be seen. (C) The structure of myofilaments (arrows) and the cytoplasmic membrane (arrowhead) of a smooth muscle cell in the tunica media is clearly visible. (D) Fibroblasts and collagen fibers in the tunica adventitia are clearly visible. (A) Bar = 500 nm; (B, C) bar = 200 nm; (D) bar = 2 μm.

Combination of LM and TEM in the Same Region of Adjacent Sections

After making serial sections, we observed endothelial cells in the same lesion using LM and TEM (Figure 4). For LM, inflammatory cells adhered to the lumen and elongated cells lining the lumen were visible (Figure 4A). The same region of the section that was observed for LM was clearly observed under TEM. Inflammatory cells and elongated cells that were observed with LM were also recognized with TEM. Additionally, macrophage-like cells were observed. Deposition of fibrinous exudation was found (Figure 4B). Macrophage-like cells had rich organelles (Figure 4C). Similarly, elongated cells that covered the lumen were also observed with LM (Figure 4D). Microvilli and cell junctions, which indicated endothelial cells, were easily identified (Figure 4E and F), while these structures were not observed under only LM. Giant cells were also observed.

OPEN IN VIEWER

Figure 4.

LM and TEM images of the endothelium in a stented artery from the same region of adjacent sections. (A) LM image of tissue around the strut. Two inflammatory cells on the lumen (arrows) and elongated cells lining the lumen (arrowhead) are visible. (B) TEM image of the same region of the section shown in (A). Two inflammatory cells on the lumen observed by LM are also recognized by TEM (arrows). Elongated cells covering the luminal surface (arrowhead) and a macrophage-like cell (white arrow) can be seen. A large amount of fibrin deposition can be seen in the tissue. (C) High-power image from (B). Sparse macrophage-like cells containing rich organelles are clearly visible (white arrow). Organelles of an inflammatory cell (arrow) and elongated cell (arrowhead) are also visible. (D) LM image of tissue around the strut. Flat-shaped cells lining the lumen (arrow) are visible. (E) TEM image of the same region of the section shown in (D). Two endothelial cells (arrows) partially overlapping and underlying a giant cell (arrowhead) can be seen. (F) High-power image from (E). Microvilli (arrowheads) and cell junctions (arrow) in the endothelium are visible. (A, D) Bar = 10 μm; (B) bar = 5 μm; (C, E) bar = 1 μm; (F) bar = 500 nm. LM = light microscopy; TEM = transmission electron microscopy.