Formaldehyde-fixed paraffin sections were cut in cross section (5 μm) and then stained with hematoxylin and eosin (HE) for analysis of structural integrity, Verhoeff's Van Gieson (EVG) staining for corruption of elastin, and Mac-3 for infiltration of macrophages.
The severity of elastin degradation is semiquantified, as previously described (grade 1, no degradation; grade 2, mild degradation; grade 3, severe degradation; and grade 4, presence of aortic rupture) [12 (link)].
Briefly, the aortic sections were deparaffinized and hydrated in H2O and then incubated with primary antibodies against Mac-3 (1 : 100 dilution, BD, 550292), MCP-1 (1 : 500 dilution, Servicebio, G11199), VCAM-1 (1 : 200 dilution, Servicebio, G13336), MMP-2 (1 : 6000 dilution, Servicebio, GB11130), and MMP-9 (1 : 1000 dilution, Servicebio, GB11132) at 4°C overnight, followed by incubation with the corresponding secondary antibody for 1 h at 37°C. Then, the sections were dehydrated and images were obtained by a Nikon microscope equipped with a CCD camera. Immunostaining data of each section was randomly selected and measured by 2 different investigators in a blinded manner using the image analysis software (Image-Pro Plus, EPIX Vision).
The severity of elastin degradation is semiquantified, as previously described (grade 1, no degradation; grade 2, mild degradation; grade 3, severe degradation; and grade 4, presence of aortic rupture) [12 (link)].
Briefly, the aortic sections were deparaffinized and hydrated in H2O and then incubated with primary antibodies against Mac-3 (1 : 100 dilution, BD, 550292), MCP-1 (1 : 500 dilution, Servicebio, G11199), VCAM-1 (1 : 200 dilution, Servicebio, G13336), MMP-2 (1 : 6000 dilution, Servicebio, GB11130), and MMP-9 (1 : 1000 dilution, Servicebio, GB11132) at 4°C overnight, followed by incubation with the corresponding secondary antibody for 1 h at 37°C. Then, the sections were dehydrated and images were obtained by a Nikon microscope equipped with a CCD camera. Immunostaining data of each section was randomly selected and measured by 2 different investigators in a blinded manner using the image analysis software (Image-Pro Plus, EPIX Vision).
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