Axioobserver fluorescence

CCGPSARAPC (GPS) peptide was purchased from Insight Biotechnology, London, UK, and was labeled with either 5(6)-carboxyfluorescein (FAM-GPS) or 5-carboxytetramethylrhodamine (TAMRA-GPS). Mice received 200 µg of peptide via tail vein injection at gestations of between E11.5 and E17.5 [7 (link)]. After 3 h, unbound peptide was removed by terminal cardiac perfusion with PBS, and maternal, placental and fetal tissues were harvested. Organs were snap-frozen in liquid nitrogen, or were fixed in paraformaldehyde (4% (w/v) in PBS, overnight), transferred to sucrose solution (30% (w/v) in PBS, 24 h), embedded in OCT (Sakura, Torrance, CA, USA) and stored at −80 °C. Snap-frozen tissues were sectioned using a cryostat, and 8 μm sections were fixed in ice-cold methanol (15 min), washed in PBS (2 × 5 min) and mounted using Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA). Peptide localization was assessed using a Zeiss AxioObserver fluorescence microscope (Zeiss, Cambridge, UK). In a separate experiment, human placental villous explant cultures were established as described below. Explants were exposed to TAMRA-GPS (0.27 µmol/L) for up to 3 h at 37 °C and were fixed and processed as above. Images were captured at the same exposure, so that comparisons of fluorescence intensity could be made between samples.