Anti crt

Cardiac broblasts (CFs) were plated on coverlips in 24-well plates. When con uent cells reached 60-70%, the cells were transfected with siRNA, then treated with Ang (10 - 5 mol/L 24hrs). Coverslips were then xed and blocked as described before [17], followed by exposure to the primary antibodies (anti-CRT 1:100 or anti-α-SMA 1:100, Cell Signaling, USA) at 4℃ overnight. After washing with PBS, the cells were incubated with uorescence-conjugated secondary antibodies for 2h at room temperature, away from light. The second antibody used was Alexa Fluor 488 Goat Anti-Rabbit IgG (1: 400, green uorescence, Invitrogen, USA) or Alexa Fluor 594 Goat Anti-Rabbit IgG(1 : 400, red uorescence, Invitrogen, USA). Cells was rinsed and incubated with diluted DAPI for 10 min, away from light. Images were collected using an Eclipse TE2000-U uorescence microscope system (Nikon, Japan) and analyzed with Image J software (NIH, USA) to semi quantitatively determine the expression of CRT and α-SMA.