Cc3 primary antibody

Single-color IHC was performed on FFPE tumor slices after six days of treatment as previously described using cleaved Caspase-3 (cC3) primary antibody (Cell Signaling Technologies, Danvers, MA, 1:200)^{35 (link)}. Whole slide imaging and digital archiving was performed using the Nanozoomer Digital Pathology system (Hamamatsu, Shizuoka, Japan), and 20 × fields (7–13 fields per group) were automatically counted using QuPath or manually counted using Fiji ImageJ Cell Counter^{36 (link)}.

Immunofluorescent detection of cleaved caspase-3 (CC3) and CD4 antigen was performed on 10-μm-thick cryo-sections mounted on glass slides (Superfrost Plus, Fischer Scientific, Pittsburgh, PA). Post-thawing at room temperature for 30 min, the tissue was fixed in cold acetone for 10 min and endogenous biotin was blocked (Avidin/Biotin Blocking Kit, Vector Laboratories, Burlingame, CA). Tissue was further blocked in a protein serum (Dako, Carpinteria, CA) for 30 min and CC3 primary antibody (1:100, Cell Signaling, Danvers, MA #9661S) was applied overnight at 4 °C. Next day, sections were washed in PBS and incubated with biotinylated rabbit secondary antibody (1:300, Vector Laboratories, Burlingame, CA) for 30 min followed by incubation with CY-3 Streptavidin (1:300, M.O.M kit, Vector Laboratories, Burlingame, CA) for 30 min. The following day, tissues were re-blocked in protein serum for 30 min and incubated overnight at 4 °C with CD4 primary antibody (1:100, Santa Cruz, Dallas, TX # sc-13573). Next day, tissues were washed in PBS and incubated with Alexa Fluor 488 Goat anti-rat secondary (1:500, Life Technologies, Waltham, MA). The tissues were counterstained with Vectastatin DAPI mounting media (Vector Laboratories, Burlingame, CA).