Borg solution

Immunohistochemistry of c-Myc was conducted by the University of Colorado Cancer Center Histology Shared Resource. Five-micron-thick paraffin sections were deparaffinized, antigen unmasked, and immunohistochemically stained for c-Myc (Abcam, Cambridge, MA; rabbit monoclonal Y69; Cat# ab32072; dilution 1:50 in TBST + 1% BSA w/v). Antigen was revealed in pH 9.5 BORG solution (Biocare Medical, Concord, CA) for 10 min at 110 °C (NxGen Decloaking chamber, Biocare) with a 10-min ambient cool down. Immunodetection was performed on the Benchmark XT autostainer (Ventana Medical Systems, Tucson, AZ) at an operating temperature of 37 °C. Primary antibody was incubated for 32 min and detected with a modified I-VIEW DAB (Ventana) detection kit. I-VIEW secondary antibody and enzyme dispensers were replaced with full-strength and half-strength (diluted in PBS, pH 7.6) polymers, respectively (Rabbit ImmPress, Vector Laboratories, Burlingame, CA: cat# MP-7401). All sections were counterstained in Harris hematoxylin for 2 min, blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in xylene, and coverglass mounted using synthetic resin. Sections were examined through bright-field microscopy and c-Myc positive regions were quantified using ImageJ software.

Tumor xenografts were harvested, fixed in 10% buffered formalin, and embedded in paraffin. Five-micrometer sections were deparaffinized, and antigen retrieval was performed in BORG solution (Biocare Medical, Concord, CA), followed by blocking with Background Sniper (Biocare Medical). Slides were incubated with anti-mouse CD45 antibody (rat IgG2b) or a control rat IgG2b antibody (BD Bioscience) and detected using the Rat on Mouse HRP detection system (Biocare Medical). Slides were then incubated with diaminobenzidine for 5 minutes and were counterstained with hematoxylin. Mouse effector cells were identified by CD45-positive staining.