Vimentin

Collected tumor tissues from dogs and xenograft were fixed in 10% buffered formalin solution (Sigma-Aldrich, St Louis, MI) and paraffin embedded for routine pathological analysis. Immunohistochemical staining was carried out on 4- to 6-μm-thick slides using an automated protocol developed for the Discovery XT automated slide staining system (Ventana Medical Systems, Inc.). Alongside haematoxylin–eosin staining (HE), the following commercially available antibodies were used for the phenotypical characterization of melanocytes: Melan-A (Thermo Fisher Scientific, Waltham, MA), S-100 protein (Dako, Agilent Technologies, Denmark), Vimentin, and cytokeratin (Biogenex Laboratories, San Ramon, CA) (see Additional file 1). Tumor sections were deparaffinized and incubated for 1 h with the appropriate antibody before incubation with Discovery UltraMap anti-Rabbit (760–4315, Roche, Basel, Switzerland) or anti-mouse horseradish peroxidase (HRP) (760–4313, Roche, Basel, Switzerland) secondary antibodies and the Discovery ChromoMap DAB kit reagents (760–159, Roche, Basel, Switzerland) (Additional file 1).

For injury marker detection, 10 µm slides from each swine in cREBOA and iREBOA groups were cut from the cortex of fresh frozen kidneys (stored in − 70 °C). Primary antibodies were diluted in 5% donkey serum and 95% primary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton + 5% bovine serum albumin). NKCC2 antibody (1:2000, Everest Biotech, EB09143) was combined with either Osteopontin (1:400 Abcam 8448, Product ID: GR5225573-4), NGAL (1:1000, BioSite, Product ID: GTX60965) or Vimentin (1:100, BioGenex, Product ID: MU074-UC). Slides were rinsed for 10 min in PBS and incubated with primary antibodies overnight at 4 °C. Then, slides were rinsed and incubated with secondary antibodies for 1 h in room temperature, and all dilutes were 1:400 in 5% swine serum and secondary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton). A mixture of Cy2 (Jackson ImmunoResearch, Product ID: 705225147) and of Cy3 (Jackson ImmunoResearch, Product ID: 715165151) was used for slides stained with Osteopontin or Vimentin. For slides stained with NGAL, a mixture of Cy2 (Jackson ImmunoResearch, Product ID: 705225147) and of Cy3 (Jackson ImmunoResearch, Product ID: 711165152) was used. Slides were then rinsed in 0.01 M PBS and mounted with Mowiol.