Ab228461

The commercial primary antibodies in this paper were as follows: anti-REPO (8D12, DSHB); anti-pH3 (Ser10) (H0412, Millipore); anti-Cleaved-Caspase 3 (9664, Cell Signaling); anti-α tubulin (ab18251, abcam); anti-MEK (8727, Cell Signaling); anti-p-MEK (Ser217/221) (3958, Cell Signaling); anti-ERK (sc-514302, Santa Cruz); anti-p-ERK (T202/Y264) (4370, Cell Signaling); anti-KCNMA1 (sc-374142, Santa Cruz); anti-Ki-67 (9129, Cell Signaling); and anti-GAPDH (ab8245, abcam).
Three BRAF-related antibodies were used in this study: anti-BRAF (sc-5284, Santa Cruz) were used to detect the total levels of BRAF; anti-BRAF^V600E specifically recognizing mutated amino acid sequence (ab228461, abcam) were used to detect the levels of BRAF^V600E; anti-p-BRAF (S729) specifically recognizing phosphorylated site (ab124794, abcam) were used to detect the levels of BRAF with serine 729 phosphorylation, to measure the BRAF activation levels regardless of the presence of BRAF mutations.

Immunohistochemical (IHC) staining for BRAF^V600E (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 BRAF^V600E and molecular methods of the detection of the BRAF^V600E 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 BRAF^V600E positivity on IHC was indicative of the BRAF^V600E 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 (CountσCell, 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 BRAF^V600E status manner.