Anti α smooth muscle actin primary antibody

Foetal heart sections were subjected to antigen retrieval in citric acid buffer (0.01 M, pH 6.0) for 12 minutes at 94°C in a microwave (BP‐111, Microwave Research & Applications). E18.5 heart sections were then stained using anti‐α‐smooth muscle actin primary antibody (1:3000; Sigma), biotinylated griffonia simplicifolia lectin‐1 (1:200; Vector Laboratories) and anti‐sex‐determining region Y protein antibody (1:200, Santa Cruz) to visualize coronary arteries, capillaries and Y chromosome, respectively. To analyse proliferation and epicardial epithelial‐to‐mesenchymal transition (EMT), E12.5 heart sections were incubated in anti‐phosphorylated histone H3 (pHH3, 1:500; Abcam) and anti‐Wilm's tumor‐1 (Wt1, 1:300; Calbiochem) primary antibodies, respectively. Primary antibodies were left on overnight at room temperature in humidity chambers. Sections were subsequently incubated for one hour at room temperature with secondary antibodies; biotinylated donkey anti‐goat IgG (1:500), biotinylated goat anti‐rabbit IgG (1:500) or biotinylated goat anti‐mouse IgG (1:500) (Vector Laboratories). Slides were imaged using the Zeiss Observer D1 microscope and AxioVision Rel 4.7 Software. All quantifications of histological images were performed using ZEN microscope software (Zeiss).13, 15, 17