Cy5621

Total proteins were extracted from colon tissue or kidney tissue samples when treated with radioimmunoprecipitation assay (RIPA) lysis (Sangon Biotech, Shanghai, China). The concentrations of total proteins were measured by bicinchoninic acid (BCA) assay (Beyotime, Shanghai, China). The proteins were separated on SDS–polyacrylamide gel electrophoresis and transferred to an NC membrane. The NC membrane was blocked with 5% non-fat milk and then incubated with primary antibodies against ZO-1 (AF5145, 1:500, Affinity), occludin (CY5997, Abways), claudin-1 (AF0127, 1:1,000, Affinity), Col-I (AF0127, 1:2,000, Affinity), Col-III (AF0136, 1:1,000, Affinity), FN (CY5621, 1:1,000, Abways), and LN (CY6617, 1:1,000, Abways) overnight at 4°C, respectively. Then, the membrane was washed with TBST and incubated with secondary goat anti-rabbit (S0001, 1:5,000, Affinity) and goat anti-rat antibodies (S0009, 1:5,000, Affinity) at 37°C for 2 h (Cell Signaling Technology, CA, USA). Finally, the proteins were monitored with enhanced chemiluminescence reagents (GE Healthcare Life Sciences, NJ, USA) and then quantitatively analyzed by ImageJ software (version 1.4.0., National Institutes of Health). GAPDH protein was used for the internal control.

The degree of renal tissue injury was evaluated by Masson’s trichrome staining. The kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4-μm paraffin sections. Then, sections were treated in xylene, dehydrated with graded ethanol, and stained with Masson (Sigma-Aldrich; Merck KGaA). After staining, the sections were dehydrated with 70 and 90% ethanol. Six fields of view were randomly selected and observed with an optical microscope (Olympus, Tokyo, Japan).
For immunohistochemistry (IHC) analysis, the renal tissues were incubated with the primary antibodies including anti-LN (CY6617, 1:100, Abways), anti-FN (CY5621, 1:100, Abways), anti-Col-I (AF0127, 1:200, Affinity), anti-Col-III (AF0136, 1:100, Affinity) overnight at 4 °C, respectively, and then further incubated with an anti-rabbit secondary antibody (ab150077, Abcam) for 2 h at room temperature. Finally, a representative area containing immunostained tissue was captured with a microscope (Olympus, Tokyo, Japan).