Mircury lna microrna ish optimization kits ffpe

Paraffin sections were air-dried at room temperature for 1.5 h and stored at 4 °C until the day before in situ hybridization, at which point they were hotplated at 60 °C for 45 min, returned to 4 °C, and used the following day. In situ hybridization was implemented according to the guidelines stated in the miRCURY LNA microRNA ISH Optimization Kits (FFPE) protocol (Exiqon, Woburn, MA). Slides were treated with Proteinase K at 15 µg/ml for 10 min at 37 °C. Hybridization was performed at 54 °C for the following: 5′-digoxigenin (DIG) labeled (U6) and double DIG (scrambled and miR-34a), LNA-modified oligonucleotide ISH probes. The following probe concentrations were used: LNA scrambled probe (40 nM), U6 (1 nM), and miR-34a (60 nM). Positive probe labeling was blue/purple. Nuclei were visualized using Nuclear Fast Red counterstain (Vector Laboratories Inc., Burlingame, CA)

Tissue microarrays were stained with rabbit antihuman Prx-6 polyclonal antibody (1:1000; Abcam, Cambridge, MA, USA). IHC staining was performed using Envision kit (Dako, Glostrup, Denmark).
The sequence of the miR-24-3p probe was 5′-CTGTTCCTGCTGAACTGAGCCA-3′ (miRCURY ™ probe, Exiqon, Vedbaek, Denmark). A U6 probe (sequence: CACGAATTTGCG TGTCATCCTT; Exiqon) was used as the positive control. ISH was implemented according to the guidelines stated in the miRCURY LNA microRNA ISH Optimization Kits (FFPE) protocol (Exiqon). Slides were treated with proteinase K at 15 µg/ml for 10 min at 37 °C. Hybridization was performed at 62 and 58 °C, respectively, for the following: 5′-digoxigenin (DIG) labeled (U6) and double DIG (miR-24-3p), LNA-modified oligonucleotide ISH probes.