Aec substrate kit

H&E and Elastic van Gieson (EVG) staining were performed frozen aortic sections to assess general morphological changes and elastin contents, respectively. To assess SMC retention, frozen aortic sections were sequentially stained with a goat anti-SMC α-actin polyclonal antibody (1:200, Cat#: NB300-978, Novus Biologicals, Centennial, CO, USA), a biotinylated rabbit anti-goat IgG antibody (1:400, Cat#: BA-5000, Vector Laboratories, Inc) and streptavidin-peroxidase conjugate (1:200, Cat#: 016-030-084, Jackson ImmunoResearch), and staining was visualized using the AEC substrate kit (Vector Laboratories, Inc). Elastin fragmentation and SMC loss were scored as the grade I (mild) to IV (severe) using a histological grading system as reported previously (26 (link)–31 (link)).

PUM spheroids were removed from the ULA plates using a cut 200-µl pipette tip at various time points and fixed in 10% neutral buffered formalin for 15 minutes. Spheroids were then suspended in 2% agar before being processed using the Bayer Tissue-Tek VIP E300 tissue processor (Bayer AG, Leverkusen, Germany). Processed spheroids were subsequently embedded in paraffin blocks and sectioned at 4 µm onto X-tra adhesive slides (Leica Biosystems, Wetzlar, Germany), for immunohistochemical (IHC) staining.
IHC staining was performed as previously described¹⁸ (link) using the Dako Pre-Treatment Module and the Dako Envision FLEX kit (Agilent Technologies, Santa Clara, CA), according to the manufacturer's instructions. Details of antibodies, antigen retrieval, and concentrations are provided in Table 1. Positive staining was visualized with an AEC substrate kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin (VWR, Leighton Buzzard, UK), dyed blue with Scott's tap water (Leica), and mounted using Aquatex aqueous mounting medium (Sigma-Aldrich). Slides were scanned using the Leica Aperio CS2 slide scanner at 20× magnification.

Frozen sections (8 μm thick) were stained by using the NoVo Link Polymer Detection System (Leica, Biosystems Newcastle Ltd, UK), followed by AEC substrate kit (Vector Laboratories Inc. Burlingame, CA), according to the manufacturer's instruction. Hematoxylin and eosin (H&E) stain and immunohistochemistry study were performed as described previously48 (link). Isotype antibody was used as the staining negative control. Anti-Ki-67 and anti-caspase 3 antibodies were from Santa Cruz and Cell Signaling.

Adult samples were fixed in 4% paraformaldehyde in 0.1 mol/L phosphate buffer overnight and decalcified with 0.5 mol/L ethylenediaminetetraacetic acid for 3 days to 1 week at 4°C. Seven micron paraffin sections were prepared. Sections were treated for antigen retrieval with 0.01 mol/L citrate buffer (pH 6.0) by microwaving for 6 min. Immunohistochemistry was performed according to Jiang et al. (1998) using the following antibodies (1:200 dilution): β‐catenin (Sigma; C2206, St Louis, MO); PCNA (Chemicon; CBL407, Billerica, MA); NCAM and tenascin‐C (Chuong and Chen 1991). Secondary antibodies were either biotinylated anti‐mouse IgG or anti‐rabbit IgG (Vector Laboratories, Burlingame, CA, USA; 1:200 dilution). The tertiary antibody was streptavidin (Vector Laboratories, 1:200 dilution). An AEC substrate kit (Vector Laboratories) was used to develop the staining. Hematoxylin was used to perform faint counterstaining. BrdU staining was performed according to Wu et al. (2004). For BrdU (BD; 347580; 1:200 dilution)/β‐catenin or PCNA/β‐catenin double staining, secondary antibodies Alexa Fluor anti‐rabbit‐488 (A11008) and anti‐mouse‐546 (A11030) from Invitrogen (Grand Island, NY) were used at a 1:200 dilution. 4’,6‐Diamidino‐2‐phenylindole (DAPI) was used to visualize the nuclei. Stained sections were imaged with a Zeiss 510 confocal microscope.

The paraffin embedded sections were cleared and the sections were incubated with 0.1% Pronase (Roche #165 921) in 0.1% CaCl2 pH 7.8. at 37C for 10 minutes. They were blocked with 3% H2O2 in TBS for 10 mins., washed then blocked with Dako Biotin Blocking System (Dako X0590). After washing, they were further blocked with 10% Normal Rabbit Serum for 10 mins at room temperature (RT) and incubated firstly with PDK1 (Abcam, ab110025) dilution 1:50, and GLUT-1 (Abcam, ab652) dilution 1: 1000 for 1h at RT, then with biotinylated Rabbit Anti-Mouse (Dako, E-0354) at 1/100 for 30 mins. at RT, and finally with Strep-ABC complex (Dako, K-0377) at 1/100 for 30 mins. at RT. The sections were developed with AEC substrate kit (vector lab, SK-4200) at RT for 20 mins., counterstained with haematoxylin and mounted with DAKO aqueous mount (Dako, 003181).