Cf660c

Paraffin-embedded 14.5 d.p.c. placenta and yolk sac sections were de-paraffinized in xylene and 100%, 95%, 80%, 70% and 50% ethanol for 3 min each and rinsed with running cold water. Antigens were retrieved in sodium citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) by microwaving for 15 min, followed by rinsing with tap water. The slides were then washed in PBS/0.025% Triton X-100 (PBST) twice for 5 min each, blocked in PBST/1.0% BSA/5.0% donkey serum for 1 h at room temperature, briefly rinsed with PBST and incubated with anti-mCherry, anti-GFP antibody (Abcam) at 4 °C overnight. The slides were washed with PBST three times for 15 min each, blocked for at least 15 min, incubated with Alexa Fluor (Life Technologies) or CF660C- (Biotium) conjugated secondary antibody for 1 h at room temperature, washed with PBST three times for 15 min each. The sections were mounted in a small drop of Vectashield with DAPI, covered with a coverslip and sealed with nail varnish. The sections were observed and imaged with a fluorescence microscope. For mCherry and Tfap2c co-staining, to reduce autofluorescence, slides were irradiated with ultraviolet light for 2 h before de-paraffining, and treated with Sudan Black for 20 min after blocking. Antibodies used are listed in Supplementary Table 7. The technical details of reducing background staining have been published³³.