Hrp dab detection kit

For histology and immunostaining, 8 µm sections were cut using a microtome (SLEE medial, Mainz, Germany). Russell-Movat pentachrome (Abcam, Cambridge, UK) or Alizarin Red staining (Sigma Aldrich, St. Louis, MO, USA) were performed according to the manufacturer’s instructions. For immunohistochemistry analysis, after antigen retrieval with EDTA buffer (Sigma-Aldrich, St. Louis, MO, USA), slices were incubated overnight with an anti-BAFF-R or CD138 antibodies (Abcam, Cambridge, UK) diluted at 1:250 or 1:8,000, respectively. Slides were stained with HRP/DAB detection kit (Abcam, Cambridge, UK) and counterstained with hematoxylin to visualize cell nuclei. Images of entire aortic valve cusps were composed using NIS-Elements Microscope Imaging Software (Nikon, Tokyo, Japan) from a series of adjacent pictures taken with a 4 × objective taken with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan). Immunostaining images were taken with a 20 × objective with a Nikon Eclipse Ni microscope (Nikon, Tokyo, Japan).

In vivo study was conducted on four male Wister rats (age: Four-week-old, body weight: 200–220 g). The animals were anesthetized with isoflurane. A dorsal shaving was performed, and the area was disinfected with an iodine solution. A 2cm incision was made in the backs of the rats, in a head-to-tail alignment orientation. The four specimens were implanted into the subcutaneous tissue of the rat. After two weeks of the implantation, the samples and surrounding tissues were retrieved from the rat. The living reaction was examined by histological evaluation, such as hematoxylin and eosin (H&E), immunostaining of vascular endothelial growth factor (VEGF) and alpha-smooth muscle actin (SMA). Antigen was detected using primary antibody, such as monoclonal rat anti-CD31 (Bio-Rad, Hercules, CA, USA) and polyclonal rabbit anti-VEGFA (Abcam, Cambridge, UK) in conjunction with an HRP/DAB detection kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. Animal experiments were approved by the Animal Care and Use Committee of Meiji University (AEFST2017-007).

Muscle samples were cut in a cryostat in 10 μm serial cross-sections, mounted on glass slides, and stained with previously characterized antibodies to demonstrate muscle fiber types and visualize the muscle cells' basement membrane. In brief, the sections were then incubated for 1 hour at room temperature with primary mouse IgM anti-MyHC I (A4.840, Developmental Studies Hybridoma Bank (DSHB) at the University of Iowa, Iowa City, IA, United States), includeed mouse IgG anti-MyHC IIA (SC-71, DSHB, and rabbit IgG anti-laminin (Z0097, Dako). Muscle sections were then washed in phosphate-buffered saline (PBS) and mounted using PermaFluor (Fisher Scientific). For visualization of capillaries, muscle cross-sections were fixed for 15 min in cold methanol at -20°C and rehydrated in PBS for 10 min at room temperature. A steamer achieved antigen retrieval using a citrate buffer (pH 6.0) for 20 min. Following quenching and protein blocking steps provided by the secondary antibody kit, slides were incubated with primary anti-CD31 antibody (ab9498) or IgG1 isotype control antibody (ab91353) from Abcam (Cambridge, United Kingdom) diluted 1:200 in PBS with 1% BSA overnight at 4°C. After three washes with PBS, secondary antibody incubation and HRP revelation were performed using the HRP/DAB Detection kit (ab64264; Abcam, Cambridge, United Kingdom) following the manufacturer’s instructions.