Ultrascan 1000 ccd

Transmission Electron Microscopy was performed on Zeiss Libra 120 Transmission Electron Microscope and images were acquired using a Gatan Ultrascan 1000 CCD. The d3 HSPCs were incubated with MPs (30 MPs/cell) in 100 µL medium for 5 hours and then fixed in 2% EM grade glutaraldehyde and 2% paraformaldehyde (Electron Microscopy Sciences, EM grade) in 0.2 M cacodylate buffer. Samples were then rinsed 3 times, 15 min each, in 0.1 M sodium cacodylate buffer containing 2 mM CaCl₂ and fixed for 1 hour in 1.5% potassium ferrocyanide, 2 mM CaCl₂, 2% OsO₄ in 0.1 M sodium cacodylate buffer on ice. After 3 washes with H₂O, the samples were post-fixed in 2% OsO₄ for 1 hour at room temperature, followed by 4 washes in H₂O. Then samples were stained en bloc overnight at 4°C with filtered 1% uranyl acetate. After 3 washes in H₂O, the samples were dehydrated in a series of ascending acetone solutions. The samples were then infiltrated within n-BGE and then Quetol-NSA resin on a rotator. Samples were embeded in labeled BEEM capsules and polymerized at 60°C for 24–48 hours. The ultrathin sections were prepared using a Reichert Jung Ultracut E ultramicrotome, and were collected onto 200 mesh formvar/carbon coated copper grids. Grids were stained with 2% methanolic uranyl acetate and Reynolds’ lead citrate.