Toluidine blue

The airway tissues were fixed with 4% paraformaldehyde for 24 h at room temperature. Then the tissues were washed overnight with tap water and dehydrated by gradient ethanol, which was prepared with different volume of 100% ethanol and double distilled water. Subsequently, the tissues were paraffin-embedded, sectioned, and dewaxed. After being stained with hematoxylin for 10 min, the tissues were further incubated with hydrochloric acid alcohol for 5 min. Then the tissues were further dyed with Masson solution (D026-1-2, Jiangcheng Bio, http://www.njjcbio.com/) for 5 min, 1% phosphomolybdic acid aqueous solution (G3472, Solarbio) for 3 min, and toluidine blue (T818873, Macklin) for 5 min at room temperature. After being soaked in gradient ethanol again for dehydration, the tissue slices were made transparent by rinsing in xylene. Finally, the tissue slices were sealed with neutral gum and the image of each section was collected under a DMLA full automatic microscope (Leica, Solms, Germany) at a magnification of 100 ×.

On reaching ~80% confluence, chondrocytes at passage 1 and passage 2 underwent morphological identification. Chondrocytes were stained using Toluidine blue (Shanghai Macklin Biochemical Co., Ltd.) and Alcian blue (Beijing Solarbio Science & Technology Co., Ltd.) for cellular identification, and were examined under a light microscope (TS100-F; Nikon Corporation). Cells were fixed with 4% paraformaldehyde in phosphate buffer. Cells were fixed at room temperature for 10–20 min, washed with running water for 3 times for 2 min and then stained. Cells were respectively stained with 1% Toluidine blue and 0.1% Alcian blue for 30 min at 37°C, the excess dye washed away with double distilled water, dehydrated with absolute ethanol and sealed with neutral gum.