Histological Evaluation of Kidney Tissue

Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3 μm-thick sections. Hematoxylin and eosin (H&E) staining was performed to assess the histological morphology. The kidney tissue section slides were incubated in Gill’s hematoxylin for 5 min, washed with tap water, incubated in 95% ethanol, and stained with eosin and phloxine for 1 min. Subsequently, the sections were dehydrated in ethanol and xylene, and were mounted with Canada balsam. For Masson’s trichrome staining, after deparaffinization with xylene, the sections were treated with Bouin’s solution at 56 °C for 30 min and were washed under running tap water until the sections were clear. The sections were subsequently stained with Weigert’s hematoxylin (A:B = 1:1), followed by staining with Biebrich Scarlet/Acid Fuchsin solution for 10 min and washing with distilled water. The sections were incubated with phosphotungstic acid/phosphomolybdic acid solution for 10 min and were treated with Aniline Blue solution for 15 min. They were subsequently incubated with acetic acid for 1 min and were dehydrated with ethanol and xylene. Collagen depositions, nuclei, and muscle fibers were stained blue, black, and red, respectively.