2.12. Histology, IHC, TUNEL, and IF.

CT26 tumor-bearing BALB/c mice (male, n = 5) were randomly assigned and i.v. injected with free drugs or NCP particles at 0.5 mg Dig/kg, 5 mg Carb/kg, and/or 50 nmol siPD-L1/mouse on a Q3D × 5 schedule. After that, mice were euthanized and gross necropsies were performed. The tissues of interest were quickly collected, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into sections for analysis. All tissues were stained with hematoxylin and eosin (H&E) before undergoing histopathological examination with an Aperio ScanScope XT Digital Slide Scanner (Leica, Germany). IHC of tumors was conducted to evaluate Caspase 3 expression. Slides were incubated with primary antibody against Caspase 3 (Novus Biologicals, NB100–56112-0.1ml, 1:1000), secondary antibody (Bethyl Laboratories, A120–101P, 1:200), and then evaluated using DAB 2 Component with Stabilizer (BioLegend) according to the manufacturer protocol. TUNEL of tumors was performed using In Situ Cell Death Detection Kit (Roche, Germany). IF of tumors was stained to visualize in vivo ICD and immune community. Slides were incubated with antibodies against CRT (Novus Biologicals, NBP1–47518AF488, 1:100), Hsp70 (Novus Biologicals, NBP1–77455AF647, 1:100), PD-L1 (Novus Biologicals, NBP1–76769R; 1:100), CD3ɛ (Novus Biologicals, NBP1–30426AF488; 1:100), CD4 (Novus Biologicals, NBP1–19371AF647, 1:100), CD3ɛ (Novus Biologicals, NBP1–30426AF647; 1:100), and CD8 (Novus Biologicals, NBP2–12183AF488; 1:100). After counterstaining with DAPI, slides were imaged with a Pannoramic MIDI II Digital Slide Scanner (3DHISTECH, Hungary).