Tunel reaction mixture

Four-week-old male BALB/c nude mice were subcutaneously implanted with HepG2 cells (1 × 10⁷ cells/150 μL/animal) to evaluate the antitumor effects of our NPs in a human-derived HCC tumour model. After the tumour sizes grew to be visible, all mice were randomly divided into four groups: PBS, CUR + RSV, NP and SP94-NP. The mice were gavaged with 10 mg/kg CUR and 50 mg/kg RSV (with equivalent doses in the NP groups) every 3 days through the tail vein. The treatment lasted 21 days, and tumour volumes and body weights were recorded regularly. Tumour volume was calculated using the following formula: tumour volume (mm³) = 0.5 × length × width². The mice were sacrificed on Day 21 posttreatment, and the frozen tumour tissue sections were incubated with a terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) reaction mixture (Roche Diagnostics) at 37 °C for 1 h. The slides were then visualized under a fluorescence microscope [57 (link)]. Additionally, routine Ki-67 immunohistochemistry was carried out according to the manufacturer’s instructions and visualized under a fluorescence microscope [57 (link)].

Apoptosis immunofluorescence staining was performed using the In situ Cell Death Detection Kit, TMR Red (Roche Applied Science, Mannheim, Germany). Initially, 10 µg/ml of proteinase K (Roche Applied Science, Mannheim, Germany) in 10 mmol/L Tris/HCl (pH 7.4) was applied to the MG specimens and left for 15 minutes at room temperature. After washing the samples with 0.1M PBS twice, TUNEL reaction mixture (Roche Applied Science, Mannheim, Germany) was added on to the samples and the label solution on the negative control samples, then incubated at 37°C for 60 minutes in a dark room. The specimens were rinsed 3 times in 1M PBS for 5 minutes each, and then 100 µl of 0.5 µg/ml DAPI diluted in Tris-buffered saline and Tween-20 were added to the samples for 5 minutes at room temperature. Finally, the specimens were washed with 1M PBS and mounted with aqueous mounting medium Permafluor (Beckman Coulter, Marseille, France). Sections were examined and photographed with an epifluorescence microscope (Axioplan2 Imaging, Carl Zeiss, Jena, Germany).

Paraffin-embedded tissue slices were dewaxed, washed with PBS and digested with proteinase K in a wet box for 15 min at 37 °C. After a repeat PBS wash, the slides were dipped in TUNEL reaction mixture (Roche Diagnostics GmbH, Mannheim, Germany) and then incubated for 1 h at 37 °C in the wet box. After washing, the sections were incubated with converter-AP for 30 min at 3 °C in the wet box, and then washed with PBS. Subsequently, the sections were stained with 3,3 diaminobenzidine, re-dyed with hematoxylin, and the signals were observed under a microscope. All cells with brown nuclei were considered to be apoptotic. Apoptotic cells were counted at a magnification of × 400 in 5 random, non-overlapping fields per section. Apoptosis index was calculated as number of apoptotic cells/number of total cells × 100%.

The prepared sections were dewaxed with xylene and then washed in an alcohol gradient. After incubation with a 0.25% Triton X-100 solution for 20 min, the sections were covered with a prepared TUNEL reaction mixture (Roche Pharmaceuticals, Switzerland) and incubated for 1 h in a dark room. Finally, each section was mounted with fluorescence decay-resistant medium and visualized under a fluorescence microscope. The data are presented as the apoptotic index (apoptotic index = the number of positive cells in each field/all cells in the field-× 100%).