Alexa flour 488 igg donkey anti mouse or anti rabbit second antibodies

The 5× 105 cells were implanted onto a cell culture dish for 24 hours (NEST Biotech, Hong Kong, China) after culturing in MRC-5-CM for 14 days. Cells were fixed with paraformaldehyde for 30 minutes, then permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature, and thereafter sealed with goat serum for 1 hour at room temperature following primary antibodies incubation in the dark for 24 hours at 4°C. Washed three times with PBS, the cells were then incubated with Alexa Flour® 488 IgG donkey anti-mouse or anti-rabbit second antibodies (1:300, Invitrogen, USA) in the dark for 1 hour at room temperature. Fluorescence images were photographed with confocal microscopy (Leica DMIRE2, Germany) (at 10×63 magnification).

5 × 10⁵ cells were implanted onto a cell culture dish for 24 h (NEST Biotech, Hong Kong, China) after culturing in MRC-5-CM for 21 days. Confocal immunofluorescent analysis: cells were fixed with paraformaldehyde for 30 min, then permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and thereafter sealed with goat serum for 1 h at room temperature following primary antibodies incubation in the dark for 24 h at 4°C. Washed three times with PBS and then cells were incubated with Alexa Flour^® 488 IgG donkey anti-mouse or anti-rabbit second antibodies (1:300, Invitrogen, USA) in the dark for 1 h at room temperature. Then, nuclei were stained with propidium iodide for 5 min. Fluorescence images were photographed with confocal microscopy (Leica DMIRE2, Germany) (at 10 × 63 magnification).