P0930

Blastocyst immunostaining was performed as described (Nichols et al., 2009 ). Briefly, embryos were washed once in PBST and once in PBS/PVP (P0930, Sigma), permeabilized for 45 min in 0.5% Triton X100 (T9284, Sigma) in PBS/PVP, blocked in 0.1% BSA with 2% donkey serum in PBST for 2h and incubated overnight with primary antibodies used at 1/100 dilution. Primary antibodies used were: Nanog (RCAB0002P-F, Cosmo Bio Co., Ltd.), Cdx2 (MU392A-UC, BioGenex), Gata6 (AF1700, R&D Systems), Gata4 (sc-1237, Santa Cruz), Sox17 (AF1924, R&D Systems). On the next day, embryos were washed in PBST and incubated for 2h with the secondary Alexa Fluor (Invitrogen) conjugated antibodies diluted 1/400 in 1% donkey serum in PBST. Blastocysts were counterstained with DAPI and mounted individually in 20 μl PBS droplets on glass-bottom Petri dishes for imaging.

Blastocyst immunostaining was performed as described previously (Nichols et al., 2009 ). Briefly, embryos were washed once in PBST and once in PBS/PVP (P0930, Sigma), permeabilized for 45 min. in 0.5% Triton X100 (T9284, Sigma) in PBS/PVP, blocked in 0.1% BSA with 2% donkey serum in PBST for 2h and incubated overnight with primary antibodies used at 1/100 dilution. Primary antibodies used were: NANOG (RCAB0002P-F, Cosmo Bio Co., Ltd.), CDX2 (MU392A-UC, BioGenex), GATA4 (sc-1237, Santa Cruz), mCHERRY (ab167453, Abcam) and GFP (A6455, Invitrogen). On the following day, embryos were washed in PBST and incubated for 2hours with secondary Alexa Fluor (Invitrogen) conjugated antibodies diluted 1/400 in 1% donkey serum in PBST. Blastocysts were counterstained with DAPI and mounted individually in 20 μl PBS droplets on glass-bottom Petri dishes for imaging.