Fast green

Whey protein isolate (WPI) (Bipro, >91% protein) was purchased from Davisco Foods International (Eden Prairie, MN, USA). CaCl₂ (≥97%) and NaCl (≥99%) were purchased from Sigma-Aldrich Merck KGaA (Darmstadt, Germany). FG was purchased from Linseed Biologic Technologies Co., Ltd. (Xinjiang, China) and used without further purification. Nile Red and Fast Green were obtained from Solarbio (Beijing, China). Sucrose, salt, corn oil, dried egg yolk, whole milk, whipped cream, jam, mayonnaise, vinaigrette and vinegar were purchased from a local supermarket (Beijing, China). Double-distilled water was used to prepare all solutions and mayonnaises. All other reagents were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and were of analytical grade.

The samples were fixed in 4% paraformaldehyde (BOSTER, China) for 72 h. After dehydration for 24 h, the samples were embedded in optimal cutting temperature compound (OCT, sakura, United States), and then performed frozen sectioning (LEICA, Germany) with a slice thickness of 8 μm. Random sections from each group were stained with HE, Safranin-O and Fast Green (Solarbio, G1371, China). Detailed operation was conducted according to the product instructions. Briefly, the frozen sections were returned to room temperature, and rinsed with tap water and then added with working solution for staining. Finally, the sections were dehydrated in alcohol and sealed with resinene. After histological staining, images were taken under a ×20 or ×40 objective with a resolution of 4,080 × 3,072. The image was taken by a light microscope (Olympus, BX53, Japan).

TMJ specimens were fixed in 4% paraformaldehyde and then decalcified in 10% EDTA for 3 months. After decalcification, the tissues were dehydrated, embedded in paraffin and sectioned (5 μm thick). The orientation of slices in rat TMJ specimens was sagittal plane orientation. The tissue sections were then stained with haematoxylin and eosin (H&E) (Solarbio), sarfarin O and fast green (Solarbio). The morphology of the cartilage and synovium were scored using the Osteoarthritis Research Society International (OARSI) and arbitrary scales, respectively.
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