Cy3 goat anti rabbit igg

MGC803 and SGC7901 cells (2,000 cells/well) cultured on collagen-coated glass coverslips were rinsed with PBS twice before fixation with 4% formaldehyde for 20 min at room temperature. The cells were rinsed then with PBS three times and permeabilized with 0.2% Triton X-100 for 10 min. To block non-specific binding, the cells were incubated with PBS containing 1% BSA for 30 min and then incubated with the primary antibody TGFβ (Cell Signaling Technology, 1:1,000) at 4 °C. Subsequently, cells were washed again and incubated with the secondary antibody Cy™ 3-Goat Anti-Rabbit IgG(Jackson, 1:100) for 2 h before staining with DAPI for 5 min. After the final wash, fluorescence microscopy (Nikon, Japan) was used to take photomicrographs of the cells. The obtained images were merged. TGFβ was stained red and the DAPI-stained nuclei appeared blue.

All rats were anesthetized using isoflurane with 3-4% induction and 1-2% maintenance. Firstly, the blood was flushed with saline through the heart, and the tissue was fixed by perfusing 250 ml 4% paraformaldehyde. We collected the tissue and dehydrated them in 20% and 30% sucrose solution in turn. After the tissues were submerged, they were frozen and continuously sliced to a thickness of 6 μm and stored at –20°C. The appropriate number of slices were randomly selected and washed in 0.01 mol/L PBS. The sections were blocked with 5% serum antibody blocking solution for 2 h at room temperature. Primary antibodies were diluted with 1% serum antibody dilution. Slices were incubated independently with Epac1 (1 : 50, Santa, USA), p120 (1 : 50, Santa, USA), CD34 (1 : 100, Abcam, UK), and CD68 (1 : 50, Abcam, UK). After incubating at 4°C overnight, sections were washed with 0.01 mol/L PBS for 10 min × 3 times. Secondary antibodies were diluted with 0.01 mol/L PBS : Cy3-goat anti-rabbit IgG (1 : 1000, Jackson, USA) and FITC-goat anti-mouse IgG (1 : 1000, Jackson, USA). The tissue sections were incubated with the corresponding secondary antibody and incubated in the dark for 2 hours at room temperature. After rinsing for 15 min × 3 times with 0.01 mol/L PBS, the sections were mounted under dark conditions. The films were photographed under a fluorescence microscope (OLYMPUS. Japan).

Cells were cultured on collagen‐coated glass coverslips for 24 hour and then rinsed with PBS twice before fixation with 4% formaldehyde for 20 minute at 37°C. Subsequently, cells were rinsed with PBS for three times and permeabilized with 0.2% Triton X‐100 for 10 minute. The cells were incubated with PBS containing 1% BSA for 30 minute and then incubated with the primary antibody at 4°C overnight. Afterwards, cells were washed and incubated with fluorophore‐conjugated secondary antibodies (Cy3™‐Goat Anti‐Rabbit IgG or Cy2™‐Goat Anti‐Mouse IgG Jackson Immunoresearch) for 2 hour and then stained with DAPI for 5 minute. After the final wash, a fluorescent microscope was used (Nikon, Japan) to collect images.