Tisues were fixed overnight in 4% paraformaldehyde, dehydrated and embedded in paraffin according to standard protocols. IHC was performed with antibodies for anti-cleaved caspase-3 (CC3) and p-Histone H3 (S10) (pHH3) (Cell Signaling Technology).
A tissue microarray (TMA), containing specimens retrieved from human MPNSTs (n=207), neurofibromas (n=56) and normal nerves (n=11) surgical resections, was used to assess phospho-PAK1/2/3 levels. Tissue sections were blocked for 20 minutes and heat-induced antigen retrieval was performed in pH 6 Citrate Buffer for 20 minutes. Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol for 10 minutes. The sections were incubated overnight with phopho-PAK1/2/3 primary antibodies (Abcam) at 4°C. Anti-rabbit secondary antibodies (Dako) were incubated for 1 h at room temperature and visualized using DAB+ chromogen (Dako). Images were captured using the Vectra Automated Quantitative Pathology Imaging System and analyzed using inForm image analysis software (PerkinElmer, Caliper Life Sciences).
All human subject studies were approved by respective institutional review boards.
A tissue microarray (TMA), containing specimens retrieved from human MPNSTs (n=207), neurofibromas (n=56) and normal nerves (n=11) surgical resections, was used to assess phospho-PAK1/2/3 levels. Tissue sections were blocked for 20 minutes and heat-induced antigen retrieval was performed in pH 6 Citrate Buffer for 20 minutes. Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol for 10 minutes. The sections were incubated overnight with phopho-PAK1/2/3 primary antibodies (Abcam) at 4°C. Anti-rabbit secondary antibodies (Dako) were incubated for 1 h at room temperature and visualized using DAB+ chromogen (Dako). Images were captured using the Vectra Automated Quantitative Pathology Imaging System and analyzed using inForm image analysis software (PerkinElmer, Caliper Life Sciences).
All human subject studies were approved by respective institutional review boards.