Mif 1

IF staining has been described in our previous reports [32 (link), 35 (link), 37 (link)]. For IHC and IF staining, rehydrated paraffin sections were first treated with 3% H₂O₂ for 30 minutes and incubated with Immuno-Block reagent (BioSB, Santa Barbara, CA, USA) for 30 minutes at room temperature. Sections were then incubated with primary antibodies specifically against CD68 (1:100, Abcam, Cambridge, MA, USA), CD14 (1:300, BioSS, Woburn, MA, USA), kidney injury molecule (KIM)-1 (1:500, R&D Systems), MIF-1 (1:100, Abcam, Cambridge, MA, USA), and Wilm's tumor suppressor gene (WT)-1 (1:1000, Abcam, Cambridge, MA, USA). Sections incubated with irrelevant antibodies served as controls. Three sections of kidney specimens were analyzed in each rat. For quantification, three randomly selected high power fields (HPFs; 400x for IHC and IF studies) were analyzed in each section. The mean number of positively-stained cells per HPF for each animal was determined across all nine HPFs.
An IHC-based scoring system was adopted for semi-quantitative analyses of podocin and WT-1 as percentage of positive cells in a blind fashion [Score of positively-stained cell for podocin and WT-1: 0 = no stain %; 1= <15%; 2 = 15∼25%; 3 = 25∼50%; 4 = 50∼75%; 5= >75%-100%/per HPF (400 x)].

Staining was performed using an automated IHC system on formalin-fixed and paraffin-embedded tissue of representative sections that were cut into 2 μm thick samples according to the protocol used in a study of the Institute of Pathology and Molecular Pathology, University Hospital Zurich, using a Ventana Benchmark XT automated staining system (Roche Tissue Diagnostics, Basel, Switzerland) [29 (link)]. The samples were loaded either in the Bond-Max system (Leica Microsystems, Wetzlar, Germany) or BenchMark Ultra system (Roche Tissue Diagnostics, Basel, Switzerland).
The following antigenes were targeted with antibodies: MIF-1 (Abcam, Cambridge, UK), CXCR2 (Thermo Fisher, Waltham, MA, USA) and CXCR4 (Abcam, Cambridge, UK). MIF-2 and CD74 antibodies were provided by the Bernhagen lab, 81377 Munich, Germany.
The staining assessment was performed according to the study of He et al. [30 (link)]; it included the Remmele score, which is calculated by multiplying the intensity and percentage of staining. Expression was considered negative or low when the score ranged from 0 to 2 and positive or strong when the score was >2.