Axiocam 503 color microscope

Histological analysis was performed on snap frozen tissue. Tissue sections were fixed in acetone or 10% neutral buffered formalin, blocked with 5% FCS/0.3% Triton-X in PBS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8, cleaved Caspase-9, p-CREB (Ser 133), p-AMPK (Thr172) (all from Cell Signaling), CD8 and MHC-I (both from BD Biosciences) followed by incubation with the appropriate secondary antibodies. Cy3-conjugated anti-rabbit secondary antibodies were used for immunofluorescence. HRP-linked anti-rabbit secondary antibodies were used for conventional staining, which were visualised with the Peroxidase Substrate (ImmPACT NovaRED). Images were taken with an Axio Observer Z1 fluorescence microscope or Axiocam 503 color microscope (ZEISS) and quantified using Image J using the fluorescence intensity (MFI) per area as previously described [72 (link)].

Histological analysis was performed on snap frozen tissue. Briefly, snap-frozen tissue sections fixed in acetone, blocked with 10% FCS and stained with anti-active Caspase 3 (BD Biosciences), cleaved Caspase 8 (Cell Signaling). For p-S6 (Cell Signaling) staining, snap-frozen tissue sections were fixed in 10% neutral buffered formalin and blocked with 5% FCS/ 0.3% Triton X-100 in PBS. Images were taken with an Axiocam 503 color microscope (ZEISS) and quantified using Image J. For conventional immunohistochemistry tumor slides, IHC profiler Image J plugin was used as previously described in detail [32 (link)].

For TUNEL staining, cells were seeded on cover slips, treated and 48 h later fixed by 4% formaldehyde in PBS for 30 min, permeabilized with 0.1% Triton X-100, 0.1% sodium citrate in PBS for 2 min and stained using the TUNEL staining kit as per manufacturer’s protocol (Roche). For p-S6 staining, cells seeded on cover slips were stained with primary anti-p-S6 antibody (Ser 235/6, Cell Signaling) overnight, followed by incubation with secondary anti-Rabbit IgG Cy3 conjugate antibody. Cover slips were incubated with DAPI in PBS for 30 min. Images were taken with an Axiocam 503 color microscope (ZEISS).