Alexa fluor 488 goat anti mouse igm μ chain

Cells were fixed as described in AP staining section. For nuclear antigen staining, the fixed colonies were treated with 0.2% Triton X-100 (Sigma) for 15 minutes. For both nuclear and cell-surface staining, colonies were exposed to blocking solution containing 5% goat serum (Sigma) and 1% BSA (Sigma) in PBS for 1 hour. The colonies were incubated overnight in blocking buffer with primary antibodies at 4 °C. After that, they were washed with PBS completely and incubated in blocking buffer with secondary antibodies for 30 minutes at RT. After incubation, they were washed with PBS and incubated with 4,6-diamidino-2-phenylindole (1:10,000) (Roche) diluted with PBS for 1 to 2 minutes. Finally, 4,6-diamidino-2-phenylindole was washed off with PBS and the colonies were observed under a Nikon fluorescence microscope.
The primary antibodies used were anti-Oct4 (C-10) (1:100, Santa Cruz), anti-Sox2 (1:400, Novus), anti-Nanog (1:400, Abcam), anti-TBX3 (1:200, Santa Cruz), and anti-TRA-1-60 (1:250, Millipore). The secondary antibodies (Invitrogen) used at 1:500 were Alexa-Fluor-488 goat anti-mouse IgM (μ-chain), Alexa-Fluor-488 goat anti-rabbit IgG (H + L), and Alexa-Fluor-594 goat anti-mouse IgG (H + L).