Quetol 651

Rapid freezing and freeze-substitution of washed cell pellets were carried out as described previously (Inokuma et al. 2020) (link). The substituted samples were transferred to -20 °C for 3 h and then warmed to 4 °C over 4 h. Next, they were dehydrated in ethanol 3 times at room temperature for 30 min each and continuously dehydrated with ethanol overnight. Infiltration was performed with propylene oxide (PO) and resin (Quetol-651; Nisshin EM Co., Tokyo Japan) at room temperature [100% PO for 30 min; 100% PO for 30 min; PO:resin 50:50 for 3 h; 100% resin overnight]. The resins were then polymerized at 60 °C for 48 h and cut into ultrathin sections of 70 nm thick using an ultramicrotome (Ultracut CUT; Leica, Vienna, Austria). The ultrathin sections were placed on copper grids, stained with 2% uranyl acetate for 15 min and lead stain solution (Sigma-Aldrich, St. Louis, MO, USA) for 3 min at room temperature. They were observed using a transmission electron microscope (JEM-1400Plus; JOEL Ltd., Tokyo Japan) at an acceleration voltage of 100 kV. Digital images (3296 × 2472 pixels) were taken with a CCD camera (EM-14830RUBY2; JOEL Ltd.).

The samples were prepared according to the protocol of Fung et al. 24 with several modifications. The cells were collected from 50 ml of LB medium (at OD 600 ~1) by centrifugation at 400 g for 10 min at RT, washed once with K-P buffer, and suspended in the same buffer. The cell suspension was then mixed with 2% (final concentration) of glutaraldehyde, and incubated at RT for 1.5 h. The cells were pelleted by centrifugation at 400 g for 10 min at RT, and then embedded in 2% agar. After the agar solidified, it was washed with K-P buffer and soaked in 1% osmium tetroxide in Veronal buffer for 1 h at RT. The agar blocks were then dehydrated by soaking in increasing concentrations of ethanol (60, 70, 80, 90 and 100% for 30 min each). The agar blocks were then soaked in propylene oxide for 30 min at RT. This step was repeated three times to completely replace the ethanol with propylene oxide. The blocks were then embedded in Quetol651 (Nisshin EM, Tokyo, Japan) and the samples were thin-sectioned (60 nm) using an ultramicrotome and a diamond knife. The samples were stained with Ti blue (Nisshin EM; diluted 10-fold) and 0.4% lead citrate. The samples were observed under a transmission electron microscope (H-7650 Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV.

After fixation, the stented arteries were dehydrated through a graded acetone series of 70%, 80%, 90%, and 2 Â 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).