Cross-sections of VGs (thickness of 10 µm) were obtained using a Microm HM-550 cryomicrotome (Carl Zeiss, Germany) and used for histological and immunohistochemical examination. Samples were stained with hematoxylin and eosin following a classic protocol (H&E; Biovitrum, St. Petersburg, Russia). A detailed protocol of immunohistochemical staining by primary monoclonal and polyclonal antibodies of type IV collagen (clone PHM-12 + CIV22), α-SMA (clone 1A4/asm-1), and factor VIII (rabbit polyclonal) conjugated with horseradish peroxidase was outlined in manuscript [26].
The thickness of neointima and calcified area were assessed quantitatively as previously described [27]. Calcified regions were determined as dark purple regions on H&E sections, as described in manuscript [28].
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