Pas stain kit

Kidney tissues were fixed in formalin, and the paraffin blocks were sliced into 4 µm sections. The kidney section was stained with PAS stain Kit (395B, Sigma-Aldrich) or H&E staining Kit (ab245880, Abcam, Cambridge, MA). Glomeruli area and mesangial matrix area were observed under a digital microscope. The mesangial matrix index was calculated by 100 × mesangial matrix area/glomeruli area. For assessing H&E staining results, the following factors were used to estimate tubulointerstitial lesions for HE staining: a, atrophy, expansion, and casts of tubules; b, tubulointerstitial fibrosis and infiltration of inflammatory cells. The specific grades varied from zero to four points: zero for no changes, one for changes of 25% or less in the section, two for changes of 25–50%, three for changes of 50–75%, and four for changes of 75–100%.

Kidneys fixed in ice-cold 4% paraformaldehyde solution were processed in an automated TP1020 tissue processor (Leica Biosystems, Wetzlar, Germany) using series of graded alcohols (Histoprep Reagent Alcohol, Cat# HC6001GAL, Fisher Scientific), SafeClear II Xylene substitute (#23044192) and molten paraffin (#4005, Tissue-Tek V.I.P processing/embedding medium, Electron Microscopy Sciences, Hatfield, PA, USA). The tissue was embedded in paraffin and 5 µm kidney sections were prepared using microtome.
For histopathological assessment of renal morphology, the sections were stained with periodic acid and Schiff’s base using PAS stain kit (# 1016460001, Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The glomerular damage was assessed by calculating the glomerular sclerotic index (GSI) in a semi-quantitative method [46 (link)]. The analysis was done by the observer blinded to treatment groups.