Sm2125

The kidney and lung tissues that were preserved in aqueous formaldehyde solution for 24 h were dehydrated and embedded in paraffin blocks. The paraffin blocks were sliced at 4-μm thickness with a sliding microtome (SM2125, Leica Biosystems, Nussloch, Germany). For histochemical examinations, the specimens were deparaffinized in xylene and sequentially rehydrated using 100% ethanol, 90% ethanol, 70% ethanol, and water. To investigate pathological features of the kidneys, periodic acid Schiff (PAS) (Sigma/Merck, Darmstadt, Germany) staining was carried out to colorize brush borders and glomerular capillaries. To examine fibrosis, the specimens were subjected to Pico Sirius-red (Merck, Darmstadt, Germany) staining. The kidney specimens were immunohistochemically stained with TGF-β1 (Arigo Laboratories, Hsinchu, Taiwan), MCP-1 (ABclonal, Woburn, MA, USA), and IL-6 (ABclonal, Woburn, MA, USA) antibodies. The distributions of TGF-β1, IL-6, and MCP-1 in the kidneys were detected using commercially available peroxidase IHC kits, and developed with diaminobenzidine (DAB) (ThermoFisher, Waltham, MA, USA). The histochemical and immunohistochemical specimens were microscopically scanned and digitalized with a Pannoramic MIDI digital scanner (3DHISTECH Ltd., Budapest, Hungary). Microscopic photos were acquired using Pannoramic Viewer 1.14 (3DHISTECH Ltd., Budapest, Hungary).

Kidney tissues were harvested and soaked in aqueous formaldehyde solution for 24 h. They were dehydrated and embedded in paraffin blocks, which were sliced to a 4 μm thickness using a sliding microtome (SM2125, Leica Biosystems, Nussloch, Germany). The specimens were stained with LMO7 and NKCC2 antibodies to validate their distribution in mouse kidneys. Immunohistochemical staining was conducted according to the manufacturers’ instructions.