Envision system hrp

An Envision two-step assay was used for the immunohistochemistry stain for Axl in WT and duplex kidney sections. Briefly, after baking at 65°C for 2 h, the sections (4 µm thickness) were dewaxed in xylene, hydrated using a graded series of alcohols (100%, 95% and 85%) and rinsed with PBS. Antigenic retrieval was performed by submerging in citric acid (pH 6.0) and microwaving. To block any nonspecific binding, the sections were treated using a 0.3% hydrogen peroxide solution for 15 min. Then, the sections were incubated overnight at 4°C using Axl antibody (1:100, CST, Danvers, MA, USA) and examined using HRP Envision Systems (Dako, Shanghai, China). Finally, the sections were visualized after counterstaining with hematoxylin. Staining results were scored semi-quantitatively based on the combined percentage (five-tiered algorithm for positive cells [0: 0%; 1: <25%; 2: 25%–50%; 3: 56%–75%; 4: >75%]) and intensity of staining (four-tiered system [0: negative; 1: weak; 2: moderate; 3: strong]), and the scores were tabulated as the expression index (percentage positive × intensity). The index scores of three pathologists were averaged to obtain the final expression index. For Axl grading, high expression was defined as a score of 2 or more; scores <2 were defined as low expression.

The livers were fixed in phosphate-buffered 10% formalin for 24 h, and paraffin sections were then prepared. Immunohistochemical staining was carried out with the EnVision/HRP system (Dako, Carpinteria, CA, USA) on deparaffinized sections treated with Target Retrieval Solution (Dako). The antibodies used were as follows: anti-insulin-like growth factor 2 (IGF2) (ab9574; Abcam, Cambridge, UK), anti-α-fetoprotein (AFP) (14550-1-AP, for mouse tissues; Proteintech Group, Chicago, IL, USA), anti-AFP (A0008, for human tissues; Dako), and anti-trefoil factor 3 (TFF3) (Abbiotec, San Diego, CA, USA). 3,3′-Diaminobenzidine tetrahydrochloride (Vector Laboratories, Burlingame, CA, USA) was used for signal detection. For the detection of the TFF3 peptide, we applied signal amplification using the TSA Plus DIG Kit (PerkinElmer, Waltham, MA, USA). In situ hybridization for non-coding H19 mRNA was carried out on deparaffinized sections using the mouse H19 QuantiGene ViewRNA Probe Set (VB6-16706; Affymetrix, Santa Clara, CA, USA) and the QuantiGene ViewRNA ISH Tissue Assay Kit (Affymetrix).