Sp2 laser scanning microscope

CF were grown to 80% confluence on 12 mm coverslips, washed with PBS, and fixed with 3.7% formaldehyde for 15 min. After the sections were pre-incubated for 30 min at room temperature in 0.3% Triton X-100 (PBS-Triton) and 10% normal donkey serum, the cells were treated with primary antibody (α-SMA and Vimentin 1:400 dilution; Collagen I, III, Fibronectin, p-ERK1/2, p-Smad2/3 1:200 dilution) overnight at 4°C and thereafter with either AlexaFluor 594 goat anti-mouse or rabbit IgG (1:1000; Invitrogen) or AlexaFluor 488 goat anti-rabbit or mouse IgG (1:1000, Invitrogen) secondary antibody for 1 hr. After extensive washing in PBS-T, cells were mounted in Fluoroshield™ with DAPI histology mounting medium from Sigma-Aldrich (MO, USA). Cells were visualized using a Leica SP2 Laser scanning microscope at the University of Chicago microscopy core facility.

Cardiac fibroblasts on 12-mm coverslips were treated with Ang or Ang
plus HKL for 72 hrs. Cells were washed with PBS, and fixed with 3.7%
formaldehyde in PBS for 15 min followed by permeabilization with 0.1% Triton
X-100 for 5 min. It is then blocked with 10% BSA in PBS followed by incubation
with primary antibody overnight at 4 °C. Thereafter cells were incubated
with secondaray antibody conjugated with either Alexa Fluor 594 or FITC for 1 h.
Cells were washed and mounted in ProLong Gold antifade reagent with DAPI. Cells
were visualized using a Leica SP2 laser scanning microscope. To quantify the
myofibroblast transformation total fluorescence of each cell (100 cells in each
group) were measured using the Image J software, and the results are presented
as relative α-SMA, collagen or fibronectin levels.