Spherical structures were also frozen using equilibrium, controlled-rate cooling after incubation in 1.5 M ethylene glycol and 0.1 M sucrose for 20-30 min. Cryovials were cooled to -70C at -20C/min, seeded and further cooled to -400C at -0.30C/min, and then cooled again to -1000C at -40C/min before storage in liquid nitrogen. Vials were thawed in a 370C water bath, and the spherical structures were washed through progressively lower concentrations of cryoprotectant media (1.5, 1.0, 0.5, and 0 M ethylene glycol) before viability analysis. amaryl diabetes medication
The procedures for ICC staining were similar to those described previously. Briefly, 2% paraformaldehyde-fixed ES cells, EBs, or their derivatives cultured on polyornithine- and laminin-coated glass coverslips were rinsed twice with PBS (5 min each rinse) and once with 0.1% Triton-X 100 in PBS (5 min at room temperature). After treatment with 10% normal serum for 20 min at room temperature, cells were incubated with primary antibodies for 24-48 h at 4°C, washed twice with PBS (5 min at room temperature for each wash), and incubated either with biotinylated (for embryonic antigens) or fluorophore-conjugated (for other protein markers) second antibodies for 1 h (in the dark for immunofluorescence). Biotinylated second antibodies were linked to the biotin/avidin system (Vectastain; Vector Laboratories, Burlingame, CA) before signal amplification with 3′,3′-diaminobenzidine (DAB; Vector Laboratories) according to the manufacturer’s protocols. After three washes in PBS (5 min each wash), cells were counterstained with 4′,6-diamindino-2-phenylindole dihydrochloride (DAPI; 300 nM; Molecular Probes, Inc., Eugene, OR), and the coverslips were mounted onto glass slides with a glycerol-based mounting solution containing 2.5% polyvinyl alcohol and 1,4-diazabicy-clo octane (Sigma). Mounted slides were placed under a hood at room temperature (for biotin/avidin/DAB) before image capture with light microscopy or in a light-proof box overnight at 4°C with storage at —20°C for immunofluorescence or confocal microscopy.