Trekkie universal link

Tissues were fixed in 10% formalin, paraffin-embedded and sectioned. The 4-µm-thick sections were used for staining. Heat-induced antigen retrieval buffers were used (citrate buffer, PH = 6, for USP5 detection; and Tris-EDTA buffer, PH = 9, for β-catenin detection) and heated for 40 min. Primary antibodies of USP5 (Santa Cruz) and β-catenin (BD biosciences) were diluted at a 1:50 dilution, respectively. Biotin-labeled secondary antibody Trekkie Universal Link (Biocare Medical, USA, STU700H) was used. Streptavidin-HRP were used to detect the antigens and visualized by 3-amino-9-ethylcarbazole (AEC) Substrate-Chromogen (ScyTek Laboratories, Logan, UT, ACD015), followed by hematoxylin counterstain.

IHC analysis was performed on the paraformaldehyde-fixed and paraffin-embedded samples. We used rabbit antibody anti-Cygb (MyBioSource and LifeSpan BioSciences) 1:1000 as the primary antibody and Trekkie Universal Link (BioCare Medical) as the secondary antibody. HRP streptavidin was used as a probe that bound to the secondary antibody. HRP was detected by DAB-Chromogen dye and visualized by ImageQuant™.

The kidney tissue was embedded in 4-μm thick paraffin sections. Then, it was deparaffinized and rehydrated using xylene and alcohol series. The specimens were stained with SR to measure the fraction area of interstitial fibrosis and with periodic acid-Schiff (PAS) to determine tubular injury. This step was followed by antigen retrieval, blocking peroxidase using 3% H₂O₂ in PBS solution, and blocking non-specific antigen using Background Sniper for immunostaining. The slides were incubated with α-SMA (1:400 dilution, Sigma, Cat. No. A2547) and CD68 (1:400 dilution, Abcam, Cat. No. ab955), PDGFR-β (1:200 dilution, Abclonal, Cat. No. A2180), MCP-1 (1:100 dilution, Abcam, Cat. No. ab25124) as the primary antibodies; TrekAvidin-HRP label, conjugated to anti-rabbit Trekkie universal Link (Biocare Medical^®), as the secondary antibody; and diaminobenzidine tetrahydrochloride (DAB). The α-SMA immunostaining was applied to measure myofibroblast expansion, and CD68 antibody was used for counting macrophage cells. The quantification was performed from 15 fields for each sample at ×400 magnification, using ImageJ software.