Phospho mtor ser2448 49f9

Pre-treatment biopsies were collected from every patient and were formalin fixed and paraffin embedded (FFPE). To determine representative tumor regions, sections of the FFPE blocks were stained with hematoxylin and eosin and assessed by a head and neck pathologist (SW). From these tumor regions, three tissue cores of 0.6 mm were obtained from the FFPE blocks and collected in a tissue microarray (TMA). The TMA was constructed using an automated tissue microarray instrument as described before [44 (link)]. TMA tissue sections (4 µm) were immunohistochemically stained with antibodies for the following antigens: phospho-mTOR (Ser2448; 49F9; 1:300; Cell Signaling), phospho-MAPK (ERK1/2) (p42/44; D13.14.4E; 1:400; Cell Signaling), PTEN (138G6; 1:100; Cell signaling). All antibodies were visualized with 3,3′-diaminobenzidine (DAB) chromagen and hematoxylin was used for counterstaining. Like TMA’s, organoids were FFPE and stained for phosphor-mTOR as described above.

Primary antibodies were incubated with the indicated dilution ratio: Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199), 1:100; Phospho-AKT (Ser473), 1:75; Phospho-mTOR (Ser2448) (49F9), 1:75; and PTEN (138G6), 1:450 (Cell Signaling Technology, Danvers, MA). After antigen retrieval (EDTA buffer pH 9 for 30 min at 95 °C), samples were incubated with primary antibody for 60 minutes at room temperature. Peroxidase-labeled anti-rabbit secondary antibody (Histofine® Simple Stain MAX PO, Nichirei Biosciences, Inc.) was then applied for 30 minutes at room temperature. Diaminobenzidine (DAB) was used for colorimetric detection. When anti p-AKT antibody was the primary antibody, samples after antigen retrieval were incubated overnight at 4 °C, followed by a 15 min incubation with peroxidase-labeled anti-rabbit secondary antibody at room temperature. Colorimetric detection was performed using the DAKO’s catalyzed signal amplification (CSA) II Biotin-free Tyramide Signal Amplification System (Agilent Technologies). Samples were deemed to have positive staining when more than 5% of cancer cells were stained.