Immunohistochemical (IHC) staining for BRAFV600E (LZ, TIR) was performed as described before (33 (link)). In brief, we used a mouse monoclonal anti-BRAF (mutated V600E) antibody (VE1) ab228461(Abcam) at a 1:100 dilution and the Novolink Polymer Detection System (250T) (Leica RE7140-K) to detect IHC reaction product. IHC staining was evaluated by three qualified observers (L.Z., T.B., T.I.R.), and full agreement was achieved; there were no specimens interpreted by any observer as potentially false-negative or false-positive. A close correlation between the results of the VE1-based IHC for BRAFV600E and molecular methods of the detection of the BRAFV600E mutation at the DNA level has been reported in a meta-analysis (35 (link)) and confirmed in our previous study using formalin-fixed paraffin-embedded material (36 (link)). Therefore, we assumed the BRAFV600E positivity on IHC was indicative of the BRAFV600E mutation.
The proliferative activity of tumors was evaluated by IHC using Ki67 antibody (clone MIB-1; DAKO, Glostrup, Denmark, 1:100 dilution) in a Ventana BenchMark ULTRA instrument. The Ki67 labeling index (Ki67 LI) was determined with the image-analyzing software (CountĪCell, Ki67 antigen Semi-Auto Counter, Seiko Tec LTD, Fukuoka, Japan) in a total of ~1,000 PTC cells (LZ). Image analysis was performed in a blind for the BRAFV600E status manner.
The proliferative activity of tumors was evaluated by IHC using Ki67 antibody (clone MIB-1; DAKO, Glostrup, Denmark, 1:100 dilution) in a Ventana BenchMark ULTRA instrument. The Ki67 labeling index (Ki67 LI) was determined with the image-analyzing software (CountĪCell, Ki67 antigen Semi-Auto Counter, Seiko Tec LTD, Fukuoka, Japan) in a total of ~1,000 PTC cells (LZ). Image analysis was performed in a blind for the BRAFV600E status manner.
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