Serial 6 μm thick cross-sections were made of the aortic sinus area on a cryotome (Reichert HistoStat, Cryostat Microtome). From the first cross-section in which the leaflets of the aortic valves appeared upward, 63 serial cross-sections were obtained, covering the entire aortic sinus area. Of every 3 consecutive cross-sections, they were subjected to anti-CD68 immunohistochemical staining, hematoxylin phloxine saffron (HPS)-staining, and Oil Red O-staining. HPS (HPS, polyscientific) and Oil Red O-staining (Fisher Scientific) were done with standard methods. Anti-CD68-staining was done with a protocol previously reported37. The sections were blocked with rabbit serum (Vectorlabs), incubated for 1 hour with rat anti-mouse CD68 primary antibody (Abdserotec, 1:250 dilution), 10 minutes with biotinylated rabbit anti-rat antibody (Vectorlabs, 1:200 dilution), thereafter 5 minutes with VECTASTAIN ABC-alkaline phosphatase solution (Vectorlabs), and finally for 17 minutes with Vector Red solution (Vectorlabs).
All cross-sections were digitally imaged with a Nikon eclipse E400 microscope, with a 10x eyepiece and 10x lenses, a Nikon DS-U1 camera box and Nikon DS-5M camera. We used NIS-Elements F3.0 software, and imaged at a 1/350s exposure time, 2560 × 1920 pixels, and pixel size of 1.46 μm2/ pix. Software written in Matlab was developed to facilitate automated image analysis.
All cross-sections were digitally imaged with a Nikon eclipse E400 microscope, with a 10x eyepiece and 10x lenses, a Nikon DS-U1 camera box and Nikon DS-5M camera. We used NIS-Elements F3.0 software, and imaged at a 1/350s exposure time, 2560 × 1920 pixels, and pixel size of 1.46 μm2/ pix. Software written in Matlab was developed to facilitate automated image analysis.