A2547

Primary smooth muscle cells were seeded, and fixed by 4% paraformaldehyde for 5 min. After washing with PBS-/- (3 times for 5 min), permeabilization with 0.3% Triton-X for 30 min, additional washing step, and blocking with 1% BSA for 30 min were performed. Incubation using primary mouse monoclonal anti-α-SM actin antibody (A 2547; Merck Millipore, Darmstadt, Germany) with a dilution of 1:400 was performed overnight at 4 °C. The next day, after a washing step, the incubation with the secondary goat anti-mouse Alexa 488 antibody (A11029, Thermo Fisher, Vienna, Austria) was performed with a dilution of 1:1000 for 1 h at room temperature. Subsequently an additional washing step and nuclei staining using propidium iodide (500 ng/µL) for 1.5 min and a final washing step was performed, followed by the mounting of the sections with ProLong Gold antifade (Thermo Fisher, Vienna, Austria). Image acquisition was performed using a Nikon Eclipse Ti microscope and NIS Elements software (Nikon Instruments, Minato, Tokyo, Japan). The analyses were performed by blinded researchers. The α-SM actin amount was determined by calculating the expression intensity in relation to the cell area.

After treatment with TGFβ, LX2 and Huh7 cells were scraped in RIPA buffer (50 mM Tris HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA, 1 mM PMSF and 5 μg/ml leupeptin) to obtain total protein extracts. Also, protein content from LX2 and Huh7 culture supernatants were concentrated using ultrafiltration units (Vivaspin Turbo 4 Ultrafiltration Unit.VS04T21, Sartorius, Thermo Fisher Scientific Inc.). Protein extracts and concentrated supernatants were boiled in Laemmli sample buffer prior to electrophoresis in 10% SDS-PAGE. Proteins were then transferred to an immunoblot nitrocellulose membrane (Bio-Rad) that was blocked with 5% non-fat dry milk and exposed to primary BMP8A antibody (ab154373 Abcam plc, Cambridge, UK), αSMA (A-2547, Merck Life Science, Darmstadt, Germany) and COL1A1 (SC-8784, Santa Cruz Biotechnology Inc., Heidelberg, Germany) antibodies overnight at 4 °C. After the incubation with the corresponding secondary antibody (Santa Cruz Biotechnology Inc.), immunoreactive bands were visualized using the ECL Western blotting protocol (Bio-Rad). Densitometric analysis of the bands was performed using Image J software. Ponceau staining was used as loading control.

Immunofluorescence (IF) staining was performed on formalin-fixed, paraffin-embedded (FFPE) sections of tumor tissues. Briefly, FFPE tissues were cut into 4 μm sections, followed by deparaffinization and rehydration using xylene and ethanol. Next, the slides were incubated in EDTA antigen retrieval buffer at subboiling temperature and then in blocking solution (BSA; G5001, Servicebio) for 30 min at room temperature. The slides were incubated overnight with primary antibodies, including anti-CD31 (ab28364, Abcam), anti-von Willebrand factor (VWF) (bs-10048R, Bioss), anti-smooth muscle actin-α (αSMA) (A2547, Merck), and anti-S100A4 (bs-3759R, Bioss) antibodies, followed by incubation with fluorochrome-conjugated secondary antibodies for 50 min at room temperature. DAPI (G1012, Servicebio) was used to stain cell nuclei. Pictures were taken with an Ortho fluorescence microscope (ECLIPSE C1, NIKON). All staining was quantified using NIH ImageJ 1.51s analysis software with the same threshold for each stain.

Four-μm-thick sections of the formalin-fixed-paraffin-embedded tissue samples were deparaffinized, incubated with 3% H₂O₂ for 10 min, underwent heat induced epitope retrieval (pH 6.0 sodium citrate buffer, 30 min in 80 °C water bath), additionally blocked with Background Block (Cell Marque, Rocklin, CA, USA) and incubated separately with mouse monoclonal primary antibodies against α-smooth muscle actin (α-SMA) (A2547, Merck, US, diluted 1:400), inducible nitric oxide synthase (iNOS) (MA5-17139, Invitrogen, US, diluted 1:400), or arginase-1 (Arg1) (ab239731, Abcam, UK, diluted 1:200) and detected by HRP-conjugated secondary goat antibodies (G-21040, Invitrogen, US, diluted 1:1000) and diaminobenzidine (DAB) with hematoxylin counterstaining.

The procedure and protocol for IHC and IF staining have been described in our previous reports [27 (link)–32 (link)]. For IHC and IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 min at room temperature. Sections were then incubated with primary antibodies specifically against zonula occludens-1 (ZO-1) (1: 200, Abcam), kidney injury molecule (KIM)-1 (1: 400, Novus), synaptopodin (1:500, Santa Cruz) and alpha-smooth muscle actin (α-SMA) (A2547, 1: 500, Sigma-Aldrich), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of kidney specimen and quadriceps muscle from each rat were analyzed. For quantification, three random chosen HPFs (200 × or 400 × for IHC and IF studies) were analyzed in each section. The mean number of positively stained cells per HPF for each animal was then determined by summation of all numbers divided by 9.
An IF-based scoring system was adopted for semi-quantitative analysis of KIM-1 in the kidney as a percentage of positive cells in a blinded fashion (score of positively stained cell for these biomarkers as: 0 = negative staining; 1 = < 15%; 2 = 16–25%; 3 = 26–50%; 4 = 51–75%; 5 = 76–100% per HPF).

Four weeks after BCP transplantation, animals were sacrificed, and their heart harvested for further histology study and protein analysis. Paraffin-embedded sections were stained with Masson’s trichrome to calculate infarct size, based on the average of 3 sections sampled at 2-mm intervals from the apex to the site of scar tissue in the LV free wall surface. For immunohistochemistry, human nuclei antibody (MAB1281, Millipore, Darmstadt, Germany), Anti-cTnT antibody (ab8295, Abcam, CB, UK), and anti-α-smooth muscle actin (A2547, Sigma, MO, USA) were used to detect cell survival and neovascularization respectively. The HNA-positive cells were calculated in 15 different fields from three different layers of sections using AxioVision Rel. 4.5 software (Zeiss, GmbH, Oberkochen, Germany).