Amacr 13h4

Immunohistochemical staining was performed using the following antibodies: CK7 (OV-TL 12/30, 1:100, DAKO, Glostrup, Denmark), CA IX (dilution 1:200, mouse monoclonal, Leica), AMACR (13H4, 1:100; DAKO, Glostrup, Denmark) and TFE3 (1:1500, Santa Cruz Biotechnology Inv., Santa Cruz, CA, USA).
Evaluation of the immunohistochemical staining was performed by light microscopy using a 10× objective lens with the selective use of a 20–40× objective lens for confirmation. The interpretation of immunoreactivity was performed in a semiquantitative manner by analyzing the extent of the staining positivity of the tumor cells. Immunostaining of greater than 10% of tumor cells was required for scoring as a positive case. The interpretation score was as follows: 0 or negative ≤10% tumor cell positivity; +1 or weak = 11–25% tumor cell positivity; +2 or moderate = 26–50% tumor cell positivity; and +3 or strong >50% tumor cell positivity [6 (link)]. Cytoplasmic and/or membranous expression of CK7 and AMACR were considered positive. Only distinct membranous staining for CA IX and distinct nuclear staining for TFE3 were considered positive [15 (link)].

Specimens, including 26 CCPRCC, 30 CCRCC and 30 PRCC, were fixed in formalin and embedded in paraffin. The 4-µm thick sections were stained with the following panel of markers: CK7 (OV-TL 12/30, 1:200; Dako, Carpinteria, CA, USA); CD10 (56C6, 1:25; Novocastra, Newcastle upon Tyne, UK); AMACR (13H4, ready-to-use; Dako); CA IX (TH22, 1:100; Novocastra, Buffalo Grove, IL, USA); vimentin (Vim 3B4, 1:250); Ki67 (MIB-1, 1:200) (both from Dako, Glostrup, Denmark). Immunoreaction was performed with an automated immunostainer from Ventana (Ventana Medical Systems, Tucson, AZ, USA). The immunohistochemistry results (CK7, C10, AMACR, CA IX and RCC maker) were interpreted as negative, weak (<30% staining), moderate (30–70% staining) and strong (>70% staining). Ki67 positive cells showed stained brownish-yellow granules in the nucleus. According to the literature written by Delahunt et al (28 (link)), the area with the highest fraction of Ki67-stained cells in section was chosen at a X10 objective magnification, then it was examined at X400 objective magnification. Finally, Ki67 labeling index (Ki67 LI) was made through counting 1,000 cancer cells (percentage of nuclei showing positive staining).