Alexa fluor 594 goat anti rabbit antibody

CFs were penetrated and incubated with the following primary antibodies at 4 °C overnight: anti-alpha smooth muscle actin antibody (α-SMA, 1:800 dilution, Abcam, ab7817, Cambridge, UK), anti-focal adhesion kinase (phospho Y397) antibody (FAK, 1:800 dilution, Abcam, ab81298, Cambridge, UK), and anti-angiotensin II type-1 receptor antibody (AT₁R, 1:100 dilution, Proteintech, 25343-1-AP, Chicago, IL, USA). Then, the cell samples were incubated with the following secondary antibodies in the dark at 37 °C for 2 h: Alexa Fluor-488 goat anti-mouse antibody (1:1000 dilution, Abcam, Ab150077, Cambridge, UK) or Alexa Fluor-594 goat anti-rabbit antibody (1:1000 dilution, Abcam, Ab150116, Cambridge, UK). Cell nuclei were stained using DAPI (1 μg mL⁻¹, Sigma, D9542, St. Louis, MO, USA). Images of the cell samples were obtained using a laser scanning confocal microscope (FV3000 Olympus, Tokyo, Japan).

Immunofluorescence staining and confocal microscopy was used to determine the trimethylated-histone H3 (Lys9) (H3K9me3) (Millipore, Billerica, MA) (1:1000). The procedures were performed as previously described^32–34 (link). The specimens were incubated for 1 h with Alexa Fluor 594 goat anti-rabbit antibody (abcam, Cambridge, UK; 1:400) after incubation of the primary antibody. Images were analysed using an A1 Nikon confocal laser scanning microscope (Nikon, Tokyo, Japan).