Cyanine3 (cy3)

FISH test was applied to examine the localization of LINC00853 and FOXP3 in GC cell lines of MGC803 and MKN-78. Briefly, Specific probes to LINC00853 and FOXP3 were prepared and labeled with cy3 (GenePharma, China) and FITC (GenePharma, China), respectively. After mixing the probes with the pre-made hybridization buffer, samples were incubated overnight in this buffer. Finally, DAPI was applied to counterstain cell nuclei. The images were captured via a confocal laser-scanning microscopy (Zeiss, Jena, Germany).

Cell migration ability was detected by transwell inserts with 8-mm-pore-size in a 24-wells format (Corning, New York, NY, USA). miR-1250-5p mimics or scramble oligonucleotides negative control labelled with Cy3 (Gene Pharma, Shanghai, China) were transfected into SH-DHL-1 cells according to the protocol previously mentioned. At 48 h post-transfection, 1 × 10⁶ transfected cells were resuspended in 100 μl of RPMI 1640 medium containing 2.5% FBS and then seeded into the upper chambers. A total of 600 μl of RPMI 1640 medium with or without SDF-1α (50 ng/ml) was added to the lower chamber. The plate was incubated at 37 °C in 5% CO₂. After 3 h of incubation, the cells labelled with Cy3 in the lower chamber were counted by Flow Cytometry (NovoCyte Quanteon™) and expressed as number of Cy3-postive cells per 200 μl. The transwell assay were repeated in three independent experiments.

Frozen brain sections were incubated with blocking buffer (5% bovine serum albumin, 0.3% PBS-Triton X-100) for 1 h at room temperature and then incubated with appropriate dilutions of primary antibodies (Table S5) overnight at 4 °C. After being washed with PBS for 3 times, brain sections were incubated with secondary immunofluorescence antibodies for 2 h at room temperature. After washed, brain sections were incubated with 1:1000 DAPI (4′, 6-diamidino-2-phenylindole) (Invitrogen; Cat. #D21490) diluted by PBS for 5 min at room temperature. The sections were then washed with PBS and covered with glass coverslips. The images were captured using a confocal microscope (Zeiss LSM710, Germany).
The locked nucleic acid-modified miR-451a probes labeled with Cy3 were designed and constructed (Shanghai GenePharma Co. Ltd), and the probe sequence was AAC+TCAGTAA+TGGTAACGGT+TT, "+" indicated the modification site of locked nucleotide. The probe signals were detected with a fluorescent in situ hybridization kit (RiboBio). In short, after pre-hybridization brain sections were incubated with miR-451a probes in hybridization solution at 37 °C overnight. The sections were washed with PBS and performed the immunofluorescent procedure described above. Two brain sections containing the mPFC or hippocampus were averaged for each mouse, and four to six mice were averaged for each group.