Hybridization buffer

Cell slides were fixed in 4% paraformaldehyde and performed in situ hybridization according to the user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon) as previous description [33 (link)]. Hybridization was performed using fluorescence-labeled KLF16 mRNA probes in hybridization buffer (Exiqon). After stringent washing with SSC buffer, slides were blocked with 10% normal goat serum. The sections were then incubated with anti-SF3B4 primary antibody (Ptoteintech, 10482-1-AP) for 1 h. After washing, the slides were incubated with a rhodamine-labeled secondary antibody (KPL, USA, 031506). Images were acquired using a Leica microscope (Leica DM6000B, Switzerland) and digitized with software of LAS V.4.4 (Leica).

miRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, hsa-miR-143, rno-miR-31, and negative controls were purchased from Exiqon (Vedbaek, Denmark). The oligonucleotides are double DIG-labeled at the 5’- and 3’-ends. ISH was performed on 6 µm FFPE sections as previously described [26 (link)-28 (link)]. Following deparaffinization, rehydration in graded alcohol and proteinase K treatment, tissue sections were hybridized with miR-143 probe (20 nM), in hybridization buffer (Exiqon) at 57°C for 14 h in a hybridizer (Dako, Glostrup, Denmark). Following stringent washes in SSC buffers, the sections were blocked against unspecific binding of the detecting antibody, using DIG wash and blocking reagent. miRNA was localized by incubation with 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-indolylphosphate (BCIP) (Roche, Mannheim, Germany). Nuclear fast red (Vector Lab., Burlingname, CA) was used as a counterstain.

In situ hybridization was performed as described previously [28 ]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), paraffin cross-sections (5-μm thick) from clinical PC tissues were deparaffinized and rehydrated for fluorescence in situ hybridization. Hybridization was performed using fluorescence-labeled miR-193a-5p probes with hybridization buffer (Exiqon) by incubation at 56 °C for 1 h in a thermo-block (Labnet, USA). After stringent washing with SSC buffer, nonspecific binding sites were blocked with 10% normal goat serum (710,027, KPL, USA). According to need, the sections were then incubated for 1 h at 37 °C with anti-HO-1 primary antibody (ab13248, Abcam) or anti-Bach2 (ab83364, Abcam) diluted 1:50 in PBS or incubated with secondary antibody directly. After washing with PBS, the sections were incubated with a rhodamine-labeled secondary antibody (031506, KPL, USA) at 37 °C for 30 min. Images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica).

In situ hybridization was performed as described previously [18 (link)]. In brief, according to user manual of miRCURY LNATM microRNA ISH Optimization Kit (Exiqon), cultured cells smears were deparaffinized and rehydrated for fluorescence in situ hybridization (FISH). Hybridization was performed using fluorescence-labeled miR-140-5p probes with hybridization buffer (Exiqon) by incubation at 56°C for 1 h in a thermo-block (Labnet, U.S.A.). After stringent washing with SSC buffer and PBS, images were acquired by using a Leica microscope (Leica DM6000B, Switzerland) and digitized with a software of LAS V.4.4 (Leica). miR-140-5p probe was purchased from GenePharma Co., Ltd (Shanghai, China). The sequence of miR-140-5p-cy3-probe was as follows: TACCATAGGGTAAAACCACTG. The in situ hybridization image analysis was done according to the protocol of FISH image analysis in clinical diagnosis. Briefly, 200 cell nuclei were (stained with DAPI) counted under fluorescence microscopy. The FISH-positive cells were stained with red probes and emitted red fluorescence. Positive-cell numbers (per 200) cells were used to statistical analysis. Each experiment was repeated three times.

Cells cultured on coverslips were fixed with 4% paraformaldehyde. Paraffin slices (4 μm) of human renal arteries were prepared, dewaxed and rehydrated for hybridization. In situ hybridization was performed using specific probes for circACTA2 under the guidance of the miRCURY LNA™ MicroRNA ISH Optimization Kit (EXIQON). The slices were hybridized with the fluorescently labeled probes by incubation in hybridization buffer (Exiqon) at 55 °C in a heat block (LabNet), and then washed with SSC buffer, and the nuclei were stained with DAPI (157,574, MB Biomedical). Images were acquired using a confocal laser microscope and digitized using LAS X (Leica) software.