Semi-thick sections (250 nm) were cut with a diamond knife on a Reichert–Jung Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany) and collected onto 100 or 200 mesh formvar–carbon-coated

AZD8055 datasheet copper grids. The grids were counterstained with saturated methanolic uranyl acetate and Reynolds’ lead citrate. The grids were coated with poly-l-lysine, and gold particles (15 or 20 nm) were absorbed to one or both sides to serve as fiducial markers for future alignment of the images of the tilt series. The sections were imaged with a Zeiss Libra 120 TEM (Carl Zeiss, Thornwood, NY, USA) equipped with a tilt stage for tomography and an in-column energy filter for enhancing contrast in the zero-loss mode. Sections were pre-irradiated to minimize specimen shrinkage during the acquisition of tomographic datasets. Tomograms were acquired from regions of the capillary walls with putative abluminal caveolae labeled with terbium as well apparently labeled free vesicles in the cytoplasm. Both single and dual axis tilt learn more series

were acquired from +60° to −60° at 1° increments using a Gatan Ultrascan 1000 2K × 2K CCD camera (Gatan, Warrendale, PA, USA). Utilizing the colloidal gold particles applied to the sections, the tilt series was reconstructed using a R-weighted back projection in IMOD 4.1 ([8]; Boulder Lab. for 3-D Electron Microscopy of Cells). The tilt series was examined with the same software. Areas of interest were selected for video analysis and computer modeling and converted to TIFF

image formats. The TIFF stacks of reconstructed tomograms were converted to a 3D data set (voxelated) with Amira 4.1.2 (Visage Imaging Inc., San Diego, CA, USA) and then thresholded using the intense electron density of the terbium precipitates to surface render vesicular compartments. Single orthoslices were translated through the Methamphetamine rendered models to ascertain the modeling accuracy of terbium deposition and its representation of vesicular compartment interiors. The models were rotated through any angle to view the most revealing perspective of the vesicular structures. Mpeg videos of these rotations and orthoslice translations were recorded to enhance the appreciation of depth and perspective. Stereovideos were also generated, which when viewed with red-cyan glasses, improved 3D viewing greatly. Terbium is a small divalent cation (130 Da), which in solution has minimal electron density. When perfused through capillaries, terbium and other lanthanides [7] bind to anionic sites in the glycocalyx on the luminal surface and membranes of vesicular compartments [16,24]. As a bound precipitate, terbium constitutes a highly electron-dense tracer that labels membranes and compartments to which it had access while in solution. When viewed in the zero-loss mode, the semi-thick sections of abdominal muscle exhibited high contrast and heavy terbium labeling of the luminal surface of the capillaries (Figure 1).