Histology and immunohistochemistry staining were performed as described recently [4 (link),5 (link)]. Briefly, mouse aortas were fixed, embedded, and sectioned at 5-μm intervals, and incubated with 3% H2O2 for 10 min. Sections were then blocked with serum and incubated with primary antibodies at 4°C overnight. Primary antibodies against Mac-2 (Santa Cruz Biotechnology, CA; 1:200 dilution), Gr-1 (Abcam, Cambridge, MA; 1:200 dilution), and IgG (Santa Cruz) were used as a negative control. Sections were then incubated with the ChemMate™ EnVision™ System (Dako, Glostrup, Denmark). Images were captured and further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i); additional images are provided in Supplementary Figure S1.
Cell apoptosis was detected using a commercial DeadEnd Fluorometric TUNEL Kit (Promega, Madison, WI). Costaining of Tunel with α-SMA or CD31 was performed to detect apoptotic SMC or apoptotic EC prior to confocal fluorescence microscopy analysis (Leica Microsystems, Buffalo Grove, IL).
HE staining was performed as described elsewhere. Elastin staining involved the use of an Elastic Fiber Staining Kit (Maixin Bio, Fuzhou, China) as described recently [5 (link)]. All images were further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i).
Cell apoptosis was detected using a commercial DeadEnd Fluorometric TUNEL Kit (Promega, Madison, WI). Costaining of Tunel with α-SMA or CD31 was performed to detect apoptotic SMC or apoptotic EC prior to confocal fluorescence microscopy analysis (Leica Microsystems, Buffalo Grove, IL).
HE staining was performed as described elsewhere. Elastin staining involved the use of an Elastic Fiber Staining Kit (Maixin Bio, Fuzhou, China) as described recently [5 (link)]. All images were further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i).
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