Immunohistochemistry

Harvested tumor samples were embedded in paraffin blocks and sectioned by the MD Anderson Research Histology Core Laboratory. Paraffin-embedded tissue samples were used to stain for Ki67 (RB-9043-P1; Thermo Fisher Scientific) and cleaved caspase-3 ([CC3]; 9661; Cell Signaling Technology). Briefly, sections of the samples were deparaffinized sequentially in xylene and decreasing concentrations of ethanol prior to rehydration and transfer to PBS. For CC3 antigen retrieval slides were placed in a vegetable steamer (Hamilton Beach) in sodium citrate (pH 6) buffer for 25 minutes. Antigen retrieval for staining for Ki67 was performed in Diva Decloaker solution (#DV2004MX; Biocare Medical). Endogenous tissue peroxidase activity was quenched with 3% hydrogen peroxide in 100% methanol for 12 minutes. Slides were then washed and blocked with 5% goat serum in PBS for 1 hour at room temperature. A primary antibody was then diluted in 5% goat serum in PBS overnight at 4°C in a humidified chamber. Ki67 was diluted at 1:200, whereas CC3 was diluted at 1:100. Slides were then washed three times with PBS. CC3 stained slides were incubated with a biotinylated anti-rabbit antibody (#GR602H; Biocare Medical) for 20 minutes at room temperature. Next, slides were washed three more times with PBS and incubated for 20 minutes with a streptavidin-horseradish peroxidase label (#HP604H; Biocare Medicare). For Ki67 staining, slides were incubated with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (#111-036-047; Jackson ImmunoResearch) diluted in 5% goat serum in PBS at a 1:500 dilution for 1 hour at room temperature. CD31 staining of frozen sections was also performed. Sections were fixed in cold acetone for 15 minutes, washed with PBS, blocked with 5% goat serum in PBS, and incubated with a rat monoclonal anti-mouse CD31 antibody (1:200, 553370; Pharmingen) overnight at 4°C. The next day, slides were washed with PBS, and an appropriate horseradish peroxidase-conjugated secondary antibody was placed on them for 1 hour at room temperature. After secondary antibody incubation, slides were again washed with PBS, briefly washed with PBS containing Brij 35 (#858366; Sigma-Aldrich), and placed in 3,3′-diaminobenzidine (#750118; Thermo Fisher Scientific). Upon color change, slides were rinsed in Milli-Q water and counterstained with hematoxylin (#GHS316; Sigma-Aldrich) for 13 seconds, rinsed in water again, and left to dry. Slides were then mounted with coverslips using Permount medium (#SP15-100; Thermo Fisher Scientific). Slides were imaged using a Leica DM4000 B LED microscope. For quantification of tumor specimens, five random high-power field (HPF) photographs of each slide were taken, and stained cells were counted manually.