Fast red substrate kit

All the mice were sacrificed 4 wk later, and the tumors were harvested separately for further experiments. The tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (HE) staining. Additionally, to investigate the possible mechanisms involved, tumor tissues from extra groups of experimental mice were harvested one week after the implantation of drug-loaded nanofilms and subjected to a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay (Abcam, England). Immunohistochemical staining was carried out with a cleaved caspase 3 antibody (CST, United States) at a dilution of 1:500 and an anti-PUMA (p53 upregulated modulator of apoptosis) (Abcam) antibody at a dilution of 1:200. Staining was performed using a Fast Red Substrate Kit (Abcam).

FFPE slides were de-paraffinized and rehydrated in graded ethanol. Antigen retrieval was initiated with a preheated 10 mM citrate buffer (pH = 6). Endogenous peroxidase was blocked with a 0.3% H₂O₂ solution (0.5 mL 30% H202 in 50 mL PBS) for 30 minutes at room temperature. The primary antibodies against CD8 (1:50, Agilent/Dako, M710301-2) and CD103 (1:200, CD103; ab129202) were diluted in phosphate buffered saline (PBS)(PBS + 1% BSA + 1% AB serum; total 80 µL) and, slides were incubated overnight at 4°C. Next, the slides were incubated with two secondary antibodies, first with envision+/HRP anti-rabbit (2 drops, K400311-2P), followed by secondary antibody immPRESS-AP mouse (MP-5402-15), both for 30 min at room temperature. For visualization, StayYellow/HRP (Abcam, ab169561) and Fast Red Substrate kit (Abcam, ab64254) were used according to manufacturers’ instructions. Appropriate washing steps with PBS, tris-buffered saline with 0,1% Tween and demi water were performed between incubation steps. Sections were mounted with Eukitt quick-hardening mounting medium (Sigma Aldrich, Steinheim, Germany), and scanned on a Hamamatsu digital slide scanner (Hamamatsu photonics, Hamamatsu, Japan). Representative staining images are depicted in supplementary Figure 3.