Samples were prepared at the Electron Microscopy Core Facility, the Ohio
State University and University of Missouri following a modified version of
National Center for Microscopy and Imaging Research (NCMIR) methods for 3D EM
[30 (link)]. Briefly, segments of suprarenal
abdominal aorta from the experimental mice (n=4) were
dissected, fixed in 4% glutaraldehyde for at least 24 h, and processed for
routine transmission electron microscopy (TEM) as previously
described1. Aortic pieces were osmium tetroxide post-fixed,
dehydrated in a graded series of ethanol, and embedded in Spurr’s epoxy
resin (Electron Microscopy Sciences). Thin sections were cut and imaged using a
JOEL JEM-1400 TEM equipped with an Olympus Veleta digital camera. To determine
the percent abnormal collagen, ImageJ (NIH) was used to trace total collagen
area and total abnormal collagen area from TEM images of remodeled adventitia.
This fraction determined for each image (n=6 per sample) was
used to determine percent abnormal collagen in each sample. Fibril diameter
measurements were determined using ImageJ (NIH) using longitudinal sections of
collagen fibrils at magnifications ranging from 25 to 50 kx. At least
n=100 fibrils were analyzed for each group by using
multiple images and TEM sections as described previously [31 (link)].
State University and University of Missouri following a modified version of
National Center for Microscopy and Imaging Research (NCMIR) methods for 3D EM
[30 (link)]. Briefly, segments of suprarenal
abdominal aorta from the experimental mice (n=4) were
dissected, fixed in 4% glutaraldehyde for at least 24 h, and processed for
routine transmission electron microscopy (TEM) as previously
described1. Aortic pieces were osmium tetroxide post-fixed,
dehydrated in a graded series of ethanol, and embedded in Spurr’s epoxy
resin (Electron Microscopy Sciences). Thin sections were cut and imaged using a
JOEL JEM-1400 TEM equipped with an Olympus Veleta digital camera. To determine
the percent abnormal collagen, ImageJ (NIH) was used to trace total collagen
area and total abnormal collagen area from TEM images of remodeled adventitia.
This fraction determined for each image (n=6 per sample) was
used to determine percent abnormal collagen in each sample. Fibril diameter
measurements were determined using ImageJ (NIH) using longitudinal sections of
collagen fibrils at magnifications ranging from 25 to 50 kx. At least
n=100 fibrils were analyzed for each group by using
multiple images and TEM sections as described previously [31 (link)].
