Mouse anti α sma

Immunohistochemical (IHC) analysis of α-SMA was performed with mouse monoclonal antibody against α-SMA. Following deparaffinization and rehydration, 0.01 M boiled citrate buffer (pH 6.0) was added into each section, and heated at 97 ± 1 °C for 10 min to achieve antigen retrieval. The sections were cooled to room temperature (RT), followed by incubation with 3% H₂O₂ (1% in 0.01 M PBS, v/v) for 10 min to remove endogenous peroxidase. After placing in PBS and blocking with 5% albumin bovine serum (Sigma-Aldrich, MO, USA) at 37 °C for 30 min, the sections were exposed to the primary antibody mouse anti-α-SMA (1:3000; Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, the sections were pre-warmed to RT for 30 min and incubated at 37 °C for 30 min. After rinsing in PBS, the sections were exposed to the secondary antibody Envision System-HRP-conjugated anti-mouse IgG (DAKO, Denmark) at 37 °C for 40 min. After the final wash, the sections were stained with 3,3′-diaminobenzidine (DAB) and recorded using a DAB-enhanced liquid substrate system (DAKO). Lastly, the sections were counterstained with hematoxylin. To verify the specificity of IHC assay, the primary antibody was replaced by Mouse (G3A1) mAb IgG1 Isotype Control (Cell Signaling Technology, MA, USA) as a negative control. The densitometry analysis of positive staining was conducted with Image-Pro Plus tool.

The cells (1 × 10⁵/mL or 3 × 10⁵/mL) were treated as above for assessing differentiation towards vascular cells and cardiomyocytes. After induction for 24 h, the medium was changed, and the cells continued to be incubated. Expression of endothelial cell marker genes CD31 and vWF (von Willebrand factor), smooth muscle cell marker genes α-SMA (α-smooth muscle actin) and CNN1, early myocardial transcription factor Nkx2.5 and GATA4 (at 1 week) and cardiac-specific genes cTnT and Cx43 (connexin-43; at 2 weeks) were analyzed with qRT-PCR. Moreover, expression of CD31, α-SMA (at 2 weeks) and cTnT (at 4 weeks) was examined with immunostaining. The cells were incubated with mouse anti-CD31 (1:100) and mouse anti-α-SMA (1:100, Abcam) or mouse anti-cTnT (1:200, Santa Cruz) overnight at 4 °C. After washing with PBS, the cells were incubated with Alexa Fluor 594-conjuncted goat anti-mouse IgG (1:400) for 30 min at 37 °C. The nuclei were counterstained with DAPI (4', 6-diamidino-2-phenylindole, 1:1000; Sigma). Expression of CD31, α-SMA and cTnT in the cells differentiated from GFP⁺ EPDCs was examined using a fluorescence microscope.

Paraffin embedded sections were deparaffinised, rehydrated in an ethanol series, permeabilised in 0.5% Triton-X/PBS and boiled in 10 mM sodium citrate for 11 mins in a pressure cooker for antigen retrieval. Samples were blocked in blocking buffer (10% normal goat serum (Sigma, G9023) in PBS-0.05% Triton-X (VWR, 28817.295). Primary antibodies were diluted in blocking buffer as follows: rabbit anti-β-catenin 1:1000 (Cell Signalling, 957 S), mouse anti-αSMA 1:100 (Abcam, ab7817), mouse anti-E-cadherin 1:500 (BD biosciences, 610182), mouse anti-Cytokeratin-18 pre-diluted (Progen Biotech 65028), rabbit anti-vimentin 1:100 (Cell signalling, 5741), rabbit anti-Cytokeratin-14 (Abcam, ab53115), mouse anti-p63 1:100 (Abcam, ab735), rabbit anti-cleaved caspase-3 1:800 (Cell signalling, 9664) and the nuclear dye Hoechst 33342 at 5 µg/ml (Thermofisher, H3570). The following secondary antibodies were all purchased from life technologies and were all diluted in blocking buffer 1:500: AlexaFluor 488 goat anti-rabbit (A11008), AlexaFluor 647 goat anti-rabbit (A21245), AlexaFluor 488 goat anti-mouse (A11001), and AlexaFluor 647 goat anti-mouse (A21237).

Wound tissues were collected on days 3, 8, and 14 after administration of wound dressings. The tissues were fixed in 4% paraformaldehyde for 24 h. Then the samples with the whole wound areas were cross-sectioned into 5 µm thick slices. Masson’s Trichrome staining (MTS), and picosirius red staining were performed. The epidermal thickness was calculated from the MTS images in the wounded region. For immunohistochemical staining, tissue sections were stained with primary antibodies including mouse anti-cytokeratin 14 (1:1000, abcam, Cat# ab7800), rabbit anti-cytokeratin 10 (1:3000, abcam, Cat# ab76318), rabbit anti-CD31 (1:50, abcam, Cat# ab28364), mouse anti-α-SMA (1:10000, abcam, Cat# ab7817) and rat anti-Ki67 (1:100, Thermofisher, Cat# MA5-14520), rabbit anti-CD86 (1:50, Cell Signaling, Cat#91882), CellROX deep red (1:500, Thermofisher, Cat#C10422), and incubated at 4 °C overnight. Alexa 647 goat anti-rabbit (1:300, Thermofisher, Cat#A-21245), Alexa 546 goat anti-mouse (1:300, Thermofisher, Cat#A-11003), Alexa 488 goat anti-rabbit secondary antibodies (1:300, Thermofisher, Cat#A-11034) were then applied. DAPI (Millipore-Sigma) was used to stain the nuclei. Images were acquired by Olympus confocal microscope and analyzed using ImageJ.

Immunofluorescence was carried out as described previously36 (link). Primary antibodies included rabbit anti-Smad4 (Santa Cruz Biotechnology), mouse anti-α-SMA (Abcam), rabbit anti-TGF-β1 (Biorbyt, Cambridge, UK), and rat anti-mouse CD147 antibodies (Abcam). Alexa Fluor 488-conjugated donkey anti-rabbit IgG(H+L) (Molecular probes), Alexa Fluor 488-conjugated goat anti-mouse IgG(H+L) Alexa Fluor 594-conjugated donkey anti-rabbit IgG(H+L), and Alexa Fluor 594-conjugate donkey anti-rat IgG(H+L) (Pierce, Rockford, USA) were used as secondary antibodies. The 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain (Molecular probes, Eugene, USA). The co-localization expression was visualized under a confocal fluorescence microscopy (Nikon, Japan).