Tcs sp5ii scanning confocal microscope

We performed in situ hybridization using locked nucleic acid (LNA)-modified DNA, which was 5′- and 3′-digoxigenin labeled. The probe was used for the detection of miR-185, U6 snRNA as the positive control, and a scramble LNA probe as the negative control. All probes were obtained from Exiqon. Hybridizations were performed on 4 µm FFPE sections of tissue with primary or distant metastases of CRC and matched paracarcinomas, following the manufacturer’s protocol, with amendments as follows: proteinase K (40 µg/ml) digestion for 30 min, 40 nM of hybridization probe, a hybridization temperature of 64 °C (Tm-30 °C), and an overnight incubation with TSA/Cy3 (Perkin Elmer) solution. For the cellular staining, an anti-DC-SIGN antibody was incubated with the blocked cells for 2 h. Cells were then incubated with a secondary antibody conjugated to FITC (ZSGB-Bio) for 1 h and mounted using DAPI (Vector Laboratories). Fluorescence signals were detected and visualized at RT using a Leica TCS SP5II scanning confocal microscope (Leica). The tissues were scored by quantifying the fluorescence density of 10 separate fields in the areas with the highest intensity of fluorescence.

For double labeling immunofluorescence analyses, cells were rinsed twice with cold PBS and fixed in 4% paraformaldehyde for 15 min at RT. The nonspecific sites were blocked by incubation with 5% BSA in PBS for 30 min at RT. Then, cells were incubated with a rabbit anti-human CEA antibody (1:100, Bioworld) diluted in PBS for overnight at 4 °C. After four washes in PBS, cells were coincubated with secondary antibodies conjugated to FITC (1:200, ZSGB-Bio) and anti-human DC-SIGN antibody conjugated to APC (eBioscience) for 1 h at RT, washed and mounted in vectashield containing DAPI (Vector Laboratories). Confocal microscopy was performed with a Leica TCS SP5II scanning confocal microscope (Leica).