Ventana dab detection kit

A Dako Omnis automated staining platform was used for immunohistochemical staining of whole tissue sections (4 μm thick) embedded in paraffin and formalin-fixed. For details regarding the antibodies used in this study, see Table 1. The antibodies were optimized using Ventana DAB detection kit (Ventana Medical Systems), and standard quality control procedures were carried out.
The immunohistochemical staining of TFE3 expressions was quantitatively scored on a scale of 0 to 3+ depending on the extent and the intensity of staining. The sample scored 0 if it did not stain at all; 1, if it stained weakly (light brown); 2, if it stained moderately intense (brown); and 3 in case of high intensity (dark brown). The percentage of TFE3-positive nuclei was scored as follows: 3 (>70%), 2 (40-70%), 1 (10-40%), and 0 (<10%). A histologic score was then generated by multiplying these two scores together, and the final positivity scores were assigned based on this histological score as follows: 3+ (a score of 9), 2+ (a score of 4 or 6), 1+ (a score of 2 or 3), and negative (a score of 0 or 1).

The 4-μm-thick sections were subjected to immunostaining with a TFE3 antibody (ZA-0657; ZSGB-BI) and a β-catenin antibody (IR702; Dako) using the Dako Omnis automated staining platform. TFE3, a monoclonal antibody was diluted to a ratio of 1:100. Animal serum and buffer were used as a negative control instead of primary antibody. Immunoreaction of TFE3 was performed on the DAKO automated immunostainer. The antibody was optimized using Ventana DAB detection kit (Ventana Medical Systems) and standard quality control procedures were carried out. The detection of TFE3 was performed following heat-induced antigen retrieval using ER2 antigen retrieval buffer. Xp11.2 renal cell carcinomas were used as positive control, while negative cell controls were also used.