iPS cells cultured in 24-well plates were washed with 500
μl/well PBS, fixed with 250 μl/well 4% paraformaldehyde
at room temperature for 20 minutes, and then washed with 500 μl/well PBS
three times. The cells were blocked and permeabilized with 250 μl/well
PBS with 0.1% Triton-X (PBS-T) supplemented with 5% bovine serum
albumin (BSA) at room temperature for 1 hour, and then in 200 μl/well
primary antibodies diluted 200 times in PBS-T with 5% BSA at 4°C
overnight. The primary antibodies (all purchased from Abcam) used in this study
were SOX2 (ab59776), OCT4 (ab19857), SSEA4 (ab16287), and TRA-1-81 (ab16289).
The cells were washed with 500 μl/well PBS-T three times, incubated with
200 μl/well secondary antibodies diluted 500 times in PBS-T with
5% BSA at room temperature for 1 hour, and then washed with 500
μl/well PBS three times. The secondary antibodies used were Alexa Fluor
488 Goat Anti-Mouse IgG (A-11001) and Alexa Fluor 488 (A-11008) or 594 (A-11012)
Goat Anti-Rabbit IgG (Life Technologies). Finally, the cells were mounted with
75 μl/well VECTASHIELD Mounting Medium with DAPI (H-1200) (Vector
Laboratories). The pictures were taken by using BZ-9000 microscope
(Keyence).
μl/well PBS, fixed with 250 μl/well 4% paraformaldehyde
at room temperature for 20 minutes, and then washed with 500 μl/well PBS
three times. The cells were blocked and permeabilized with 250 μl/well
PBS with 0.1% Triton-X (PBS-T) supplemented with 5% bovine serum
albumin (BSA) at room temperature for 1 hour, and then in 200 μl/well
primary antibodies diluted 200 times in PBS-T with 5% BSA at 4°C
overnight. The primary antibodies (all purchased from Abcam) used in this study
were SOX2 (ab59776), OCT4 (ab19857), SSEA4 (ab16287), and TRA-1-81 (ab16289).
The cells were washed with 500 μl/well PBS-T three times, incubated with
200 μl/well secondary antibodies diluted 500 times in PBS-T with
5% BSA at room temperature for 1 hour, and then washed with 500
μl/well PBS three times. The secondary antibodies used were Alexa Fluor
488 Goat Anti-Mouse IgG (A-11001) and Alexa Fluor 488 (A-11008) or 594 (A-11012)
Goat Anti-Rabbit IgG (Life Technologies). Finally, the cells were mounted with
75 μl/well VECTASHIELD Mounting Medium with DAPI (H-1200) (Vector
Laboratories). The pictures were taken by using BZ-9000 microscope
(Keyence).