Optical microscopy

Kidney paraffin-embedded sections were stained using haematoxiylin-eosin following the standard protocol. Subsequently, two independent, blinded to treatment researchers observed the kidneys with the optical microscopy (Olympus, Hamburg, Germany) at a final magnification of 400×. Kidney-embedded paraffin sections were stained with haematoxylin–eosin, according to standard protocol and then were observed with an optical microscopy (Olympus, Hamburg, Germany) at a final magnification of 400×. Kidney injury scores were assessed analyzing glomerular and tubular morphology, inflammatory cells infiltration, glomerular hypercellularity, proximal tubules brush border detachment, tubular lumen narrowing and epithelial tubules alterations. Further details about kidney injury scores are reported by Dos Santos et al. [26 (link)].

For immunohistochemistry, cells were plated on gelatinized cover slips in 35-mm petri dish. At about 90%–100% confluence, cell on cover slips were washed, fixed, blocked, and then primary antibody was added and incubated for 12 h at 4°C. After the washes, the secondary antibody was added at 37°C for 1 h. The stained sections were examined and imagined under confocal (Leica Microsystems GmbH, Mannheim, Germany) or optical microscopy (Olympus Optical Co. Ltd, Tokyo, Japan).

The specimens were fixed in 10% buffered formalin (Merck, Darmstadt, Germany) for 24 h, followed by dehydration in a graded series of ethanol and embedding in paraffin. In the transverse axis to the implant, thin sections (5 μm) were prepared using a microtome (Leica Microsystems SP 1600, Nussloch, Germany). The specimens were stained with hematoxylin and eosin (H.E. stain, Merck) and examined using optical microscopy (Olympus Optical Co., Tokyo, Japan).