Tunel fluorescent kit

Freshly cut FFPE sections were evaluated by immunofluorescent staining, as previously described.^{6 (link)} The following primary antibodies were used: anti-GRP78 (C-20; Santa Cruz Biotechnology; 1:50), anti-phospho-AKT (Ser473) (D9E XP; Cell Signaling Technology; 1:50), anti-phospho-S6 (Ser235/236, Cell Signaling; 1:100), and anti-S6 (Cell Signaling; 1:100). DAPI was used for nuclear staining. Immunofluorescence was analyzed by Zeiss LSM 510 confocal microscope with LSM 510 Version 4.2 SP1 acquisition software. Confocal images were acquired with 40X and 100X oil lens, and processed with LSM Image Browser R4.2 and Adobe Photoshop CS5.
Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) fluorescent kit (Promega, Madison, WI) on FFPE sections, according to manufacturer’s instructions, as previously described.^{44 (link)} The percentage of TUNEL-positive cells was determined using ImageJ.