Toluidine blue staining

The medium were changed every two days, and the mast cells were detected using Toluidine blue staining. Following 4 weeks of culture, 0.5 ml cell suspension was collected with a Pasteur tube, added dropwise onto an autoclaved cover glass covered with polylysine and then air-dried. Cells were stained with toluidine blue for 15 min at room temperature, washed with water, followed by acetone differentiation, gradual ethanol (95, 85 and 75%) dehydration, xylene hyalinization and neutral resin mounting at room temperature. Slides were imaged using a microscope (CX41; Olympus Corporation; magnification, x200).