Fv 1000 laser scanning confocal biological microscope

For IF study, MALAT1 knockdown and control cells were incubated with primary antibodies (1:200 dilutions) overnight at 4  °C, followed by incubated with FITC-labeled secondary antibody (1:100 dilutions, Santa Cruz, CA) for 1 h at room temperature. Nuclei were counterstained with DAPI reagent (Life technology, USA). Positive cells were visualized using FV-1000 laser scanning confocal biological microscopes (Olympus, Japan).

The cell apoptosis of the tumor samples was measured by TUNEL assay using an in situ cell death kit (Roche) by following the manufacture's protocol. The cell nucleuses were counterstained by DAPI (Sigma) reagent. Positive cells were visualized by FV-1000 laser scanning confocal biological microscopes (Olympus).

All animal experimental protocols were approved by Tianjin Medical University Animal Care and Use Committee. The NU/NU nude mice at age of 4 weeks, female, SPF grade (Vital River Laboratories, China) (n = 50, 10 for each group) were implanted subcutaneously with 5 × 10⁶ Tca8113/DDP cells as described previously36 (link). The tumor volume was measured with a caliper every 3 days using a formula (volume = long diameter × short diameter²/2). When the volume of xenograft tumors was approximately 250 mm³, the tumors is injected with DMSO, DDP (7.5 mg/kg, local injection), WP1066 (40 mg/kg, local injection), and WP1066 combined DDP, every 3 days. After 3 weeks, the mice were sacrificed and the xenograft tumors were removed for formalin fixation and preparation of paraffin-embedded sections.
Apoptosis (programmed cell death) in the tumor specimens of the murine model was examined by TUNEL method using an in situ cell death kit (Roche, USA) according to the manufacture's protocol. Nuclei were counterstained with DAPI reagent (Life technology, USA). Positive cells were visualized using FV-1000 laser scanning confocal biological microscopes (Olympus, Japan).