Explanted EJV segments were perfusion-fixed in formalin. Paraffin-embedded tissue cross-sections from the center of the venous anastomosis were stained with elastic Van Gieson's stain (Accustain Elastic Stain, Sigma). The NH index was calculated as the ratio of the NH area to the combined area of the graft and media, as previously described (25 (link)). Tissue sections from the same anastomotic region were also immunostained for the nuclear proliferation marker, Ki-67, and for the EC marker, von Willebrand factor (vWF). The proliferation index (PI) was calculated as the ratio of the number of Ki-67-positive cells relative to the number of all cells in the same area, as described previously (6 (link)). Direct manual counting of the vWF-positive neovessels in the NH and the adventitia in a given image field (i.e., neovessel density) was performed separately by three independent observers under light microscopy at a magnification of ×20. Tissue sections from the same anastomotic region were also subjected to immunohistochemistry using antibodies against VEGF, VEGFR-2, PDGF-B and PDGFR-α.
Accustain elastic stain
Manufactured by Merck Group
Accustain Elastic Stain is a laboratory reagent used for staining and visualizing elastic fibers in histological samples. It provides a consistent and reliable method for the identification and analysis of elastic components in tissues.
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3 protocols using accustain elastic stain
1
Tissue Analysis of Venous Anastomosis
2
Histological Characterization of Aortic Structure
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Aortas were perfused at constant pressure with phosphate buffered saline + 1 mM EDTA. The heart and 3 cm of aorta were then removed, cleaned, placed in formalin, and processed into paraffin blocks. Paraffin sections (5 μm) of the thoracic aorta were deparaffinized, fixed, and stained with H&E, Mason’s Trichrome, Accustain Elastic Stain (Sigma-Aldrich, St. Louis, MO), or Picro-sirius Red Stain (ScyTech, Logan, Utah) kits. In selected studies, frozen sections from the aorta were stained with Oil Red O (American Mastertech).
3
Partial Carotid Ligation Model for Atherosclerosis
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Partial carotid ligation was performed as described before (Nam et al., 2009 (link)). In brief, mice at 12 wk of age were anaesthetized by intraperitoneal injection of ketamine (120 mg/kg, Pfizer) and xylazine (16 mg/kg, Bayer) and placed on a heated surgical pad. After hair removal, a midline cervical incision was made, and the left internal and external carotid arteries were exposed and partially ligated with 6.0 silk sutures (Serag-Wiessner), leaving the superior thyroid artery intact. Skin was sutured with absorbable 6.0 silk suture (CatGut) and animals were monitored until recovery in a chamber on a heating pad after the surgery. Animals were fed a high-fat diet for 2 or 4 wk (Ssniff, TD88137), at which time their carotid arteries were harvested. To determine atherosclerotic lesions, left (partially ligated) and right (sham) carotid arteries were removed and fixed in 4% PFA overnight. Fixed vessels were embedded in paraffin. Serial sections (5 µm) were made through the entire carotid arteries and stained with accustain elastic stain (Sigma) according to manufacturer’s instructions. Plaque area was calculated by subtracting the lumen area from the area circumscribed by the internal elastic lamina.
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