Cy3 conjugated goat anti rabbit igg h l

Immunofluorescence assays (IFAs) were carried out as previously described [70 (link)]. Briefly, HFFs infected with tachyzoites were fixed with 4% formaldehyde, permeabilized with 0.25% Triton X-100 (Solarbio, Beijing, China) in phosphate-buffered saline (PBS, Solarbio, Beijing, China), and blocked with 3% bovine serum albumin (BSA, M&C Gene, Beijing, China) in PBS. Cells were then incubated with primary antibody diluted in 3% BSA/PBS for 1 h at 37 °C. After washing with PBS, the cells were incubated with secondary antibodies (FITC-conjugated goat anti-mouse IgG (H + L), 1:100, or Cy3-conjugated goat anti-rabbit IgG (H + L), 1:100, Proteintech, Wuhan, China) for 1 h at 37 °C. Finally, cells were sealed with an antifade mounting medium containing DAPI (Solarbio, Beijing, China) and images were captured using a fluorescence microscope (Olympus, Tokyo, Japan).

The HDPCs (5 × 10⁴ cells) were seeded in the 35 mm laser confocal dishes for 24 h and starved in the serum-free medium for 24 h. Then they were randomly divided into four groups, which were the control group, the D-gel group, the D-gel+U0126 group and the U0126 group. The cells were first treated with 10 µM p-ERK1/2 inhibitor U0126 in the D-gel+U0126 group and the U0126 group for 1 h. Next, 1 µM D-gel diluted by serum-free medium was added to stimulate cells in the D-gel group and the D-gel+U0126 group for 1 h. The cells were fixed in 4% cold formaldehyde for 15 min. Then the cells were covered with 100% cold methanol and incubated at –20°C for 10 min. After washed with PBS for 5 min and blocked with blocking buffer (Beyotime, Shanghai, China) at room temperature for 10 min, the cells were incubated with a 1:200 dilution of anti-rabbit ERK1/2 (Beyotime, Shanghai, China) and anti-rabbit p-ERK1/2(Cell Signaling Technology, USA) at 4°C overnight. A 1:200 dilution of 488-conjugated goat anti-rabbit IgG (H + L) or Cy3 conjugated goat anti-rabbit IgG (H + L) (Proteintech Group, USA) was used as the secondary antibody for 1 h. The cell nucleus was stained with DAPI for 5 min (Beyotime, Shanghai, China). The images were captured using a confocal microscope (Leica, Germany).