Isoamyl acetate

Wild-type and Dyrk2^-/- embryos at E10.5 were washed with 0.1 M phosphate buffer (PB) (pH7.5) and fixed with 2% glutaraldehyde (TAAB Laboratories Equipment, Berkshire, England) in 0.1 M PB (pH 7.4) for 1 week at 4°C. The embryos were placed in tannic acid in 0.1 M PB for 2 hr at room temperature in darkness, and then immersed in 1% OsO₄ solution for 2 hr at room temperature. After dehydration in graded ethanol, the samples were transferred into isoamyl acetate and dried at the critical point in liquid CO₂, and this was followed by a metal coating procedure (Hitachi, Tokyo, Japan). The surfaces of tissues were then observed using scanning electron microscopy (Hitachi).

To observe microstructure, the bone marrow tissues were taken by the aforementioned method from each group of rats (n=10) on days 0 and 7. The tissues were first fixed with 4% glutaraldehyde (Shanghai Yuanye Biotechnology Co. Ltd, Shanghai, China) for more than 4 h, then were washed three times with the PBS, post-fixed with 1% potassium phosphate-buffered osmium tetroxide (pH = 7.0, provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) for 1 h, and washed three times with PBS. Then, the tissues were dehydrated using a graded series of ethanol (30, 50, 70, 80, 90, 95, and 100%) for 15–20 min at each step, transferred to the mixture of alcohol and isoamyl acetate (provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) for approximately 30 min, and then transferred to pure isoamyl acetate for approximately 1 h. Finally, the tissues were dehydrated in a critical point dryer (Hitachi, Japan) with liquid CO₂. The dehydrated tissues were coated with gold-palladium (provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) and observed under a scanning electron microscope (SEM; Hitachi, Japan).