Cells were resuspended in 2.5 mL fixative solution and fixed at room temperature for 20 min. Samples were centrifuged; cell pellets were resuspended in 0.5 mL fixative and chilled on ice for 1 hr.
The coverslips were washed 4 times with Hank's buffered salt solution and fixed with freshly made fixative (methanol-glacial acetic acid, 3 1, v/v) for 5 min, followed by air-drying and 15 min Giemsa stain.
At the time of harvesting, the cells were detached, swollen in hypotonic solution, and fixed in cold modified Carnoy's fixative.
Significantly, Hsd showed selectively response to RNA in aqueous solution and fixed cells.
For further fixation the brain hemispheres were transferred to 20%% sucrose solution and fixed overnight at 4 °C.
The root tips were removed from this solution and fixed in Carnoy's solution I (ethanol:acetic acid = 3 1) (Sharma and Sharma 1965) at 5 °C overnight.
Both algorithms have been shown to converge to the Stackelberg equilibrium while providing substantial gain compared to optimal solution and fixed full reuse scheme.
The suspensions were then mixed with 1ml of 4% (w/v) freshly prepared paraformaldehyde solution and fixed for 1 h at room temperature.
At considered time points for morphological investigation (4 h, 1, 3, 5, and 7 days), cell cultured samples were detached, washed with PBS solution and fixed in 4% glutaraldehyde solution for 30 min.
Briefly, the scaffolds were washed once with PBS solution and fixed with 2.5% glutardialdehyde (75% solution in water, Sigma-Aldrich, Germany) buffered with 0.1 M cacodylate (Sigma-Aldrich, Germany), pH 7.4, for 2 h at 4 °C.
