α smooth muscle actin α sma

After euthanasia, thoracotomy was performed to remove the heart and proximal aorta, which were frozen in optimal cutting temperature compound (10.24% polyvinyl alcohol, 4.26% polyethylene glycol, and 85.5% nonreactive ingredients). The apical two thirds of the heart was removed, and remaining tissue was secured in a cryostat. Sections, 10 μm thick, were obtained from proximal, mid, and distal aortic valve from each mouse. Slides were stained with Alizarin Red or Picrosirius Red to quantitate the amount of calcium or collagen, respectively, as described previously.12 Movat's Pentachrome stain was used to visualize proteoglycans and collagen in the aortic valve, as described previously.13 Sources of primary antibodies for immunostaining were as follows: collagen‐1 (Abcam, Cambridge, MA), α‐smooth muscle actin (α‐SMA; Abcam), Runx2 (Santa Cruz Biotechnology, Dallas, TX), and CD31 (BD Biosciences, San Jose, CA).
To detect phosphorylated EGFR in aortic valve, hearts from control mice and Velvet mice, aged 2.5 to 4 months, were incubated in DMEM in the presence (N=5 control, and N=6 Velvet) or absence (N=5 control, and N=5 Velvet) of recombinant EGF (40 ng/mL; R&D Systems, Minneapolis, MN), for 12 minutes, then frozen in optimal cutting temperature compound, cut in 10‐μm sections, and stained with anti–phosphorylated EGFR antibody (Cell Signaling, Danvers, MA).

Cancer‐associated fibroblasts and NFs were separated and purified by brief exposure to 0.25% trypsin‐EDTA (Invitrogen). Representative images were recorded using a phase‐contrast microscope. Immunocytochemistry was used to verify the identities of CAFs and NFs. The CAFs and NFs cells suspended in BEGM were plated on glass coverslips. The cells were then incubated at 37°C for 24 h and immersed in 4% paraformaldehyde (PFA) for 15 min, washed with PBS and incubated in 10% normal goat serum (Boster) for 40 min to block nonspecific interactions, in the presence or absence of 0.3% Triton X‐100 to permeabilise the cells. The coverslips were immersed in solutions containing primary antibodies (rabbit antihuman) for pan‐cytokeratin (CK; 1:400; Abcam), vimentin (1:200; Abcam), α‐smooth muscle actin (α‐SMA; 1:200; Abcam) and fibroblast activation protein (FAP; 1:250; Abcam) and incubated at 4°C overnight. The cells were then treated with secondary antibodies (Jackson ImmunoResearch), either fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit IgG (H + L; 1:100) or Cy™3‐conjugated goat anti‐rabbit IgG (1:100) for 1 h at 37°C. The cell nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Boster).

The following primary antibodies were used: neuron-specific β-III tubulin (diluted 1:200, Abcam, Cambridge, UK), neuronal nitric oxide synthase (nNOS, diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), α-smooth muscle actin (α-SMA, diluted 1:500; Abcam), VEGF (diluted 1:200; Santa Cruz Biotechnologies) and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).