All the kidney tissues were excised, fixed in 4% formaldehyde and embedded in paraffin. Kidney tissue sections (5 μm) were obtained and used for histological and immunohistochemical analyses. H&E staining was performed according to the standard H&E protocol. The injury score was acquired by modified Banff classification criteria in ten randomly selected non-overlapping fields per rat H&E stained kidney tissues59 (link). All specimens were blindly evaluated by one nephrologist.
For renal fibrosis, Masson’s trichrome staining from the each group was used to evaluate the extent of renal fibrosis according to the standard Masson’s trichrome protocol. Briefly, kidney tissue sections were successively immersed into Weigert’s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. To quantify the renal fibrosis, the blue pixel contents of the images were photographed with the same microscope and magnification times. Ten different views in each group were selected to detect the values of the integral optical density and the total area and the expression intensity was calculated as the percentage of the integral optical density to the total area which was performed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.).
For renal fibrosis, Masson’s trichrome staining from the each group was used to evaluate the extent of renal fibrosis according to the standard Masson’s trichrome protocol. Briefly, kidney tissue sections were successively immersed into Weigert’s iron hematoxylin, Biebrich scarlet-acid fuchsin, phosphomolybdic-phosphotungstic acid, and aniline blue. To quantify the renal fibrosis, the blue pixel contents of the images were photographed with the same microscope and magnification times. Ten different views in each group were selected to detect the values of the integral optical density and the total area and the expression intensity was calculated as the percentage of the integral optical density to the total area which was performed by Image-Pro Plus 6.0 (Media Cybernetics, Inc.).
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