Aniline blue

Renal pathology was evaluated via hematoxylin and eosin (H&E), Masson and PAS staining. Mouse kidney tissue was embedded in paraffin. For H&E staining, after deparaffinization and dehydration, cell nuclear and cytoplasm of kidney sections were respectively labeled by hematoxylin and eosin according to manufacture`s introduction (Servicebio, China). For Masson staining, renal sections were stained with Lixin red, phosphomolybdic acid treatment, and aniline blue according to manufacture`s introduction (Servicebio, China). For PAS staining, the sections were oxidized with periodic acid and then stained with Schiff reagent (Servicebio, China).

To assess collagen production in all groups, Masson staining was carried out. The tissue sections were deparaffinized and dehydrated and stained using hematoxylin (G1004; ServiceBio Inc., Boston, MA, United States), differentiated with hydrochloric acid-alcohol for 5 s and stained with a ponceau-acid fuchsin (G2011; ServiceBio Inc., Boston, MA, United States) for 5 min. The stained sections were placed in 1% phosphomolybdic acid for 2 min and stained with aniline blue (G1071; ServiceBio Inc., Boston, MA, United States) for 5 min and subsequently dehydrated and sealed with neutral gum (G1403; ServiceBio Inc., Boston, MA, United States). The stained sections were photographed by the microscope and the collagen expression level was analyzed using Image-Pro Plus software.

Masson staining was employed to observe the collagen production and distribution at the contact surface between the screw and bone tissue. After sectioning as same as Hematoxylin-eosin staining section, the sections were dyed with hematoxylin dye, redyed with Masson Deli Chunhong acid reddish Staining Solution (Solarbio, China), immersed and differentiated in 2% glacial acetic acid (Servicebio, China) and 1% phosphomolybdic acid (Servicebio, China), then stained with aniline blue (Servicebio, China), leached by 2% glacial acetic acid, dehydrated by ethanol and hyalinized by xylene, finally sealed with neutral gum and observed under an inverted fluorescence microscope.