Tsatm biotin system

To detect and localize miR‐155, we exploited the higher specificity and hybridization efficiency of locked nucleic acid (LNA) probes in frozen human NP tissues.23, 24 LNA hybridization probes labeled with digoxigenin (DIG) complementary to human mature miR‐155 and negative controls (scramble probes) were purchased from Exiqon (Copenhagen, Denmark). The miR‐155 sequences are given at http://www.microrna.org. In situ hybridization (ISH) reactions were performed as described previously.23, 25 In brief, slides were subjected to proteinase‐K digestion for 10 min and subsequently prehybridization buffer for 4 h. LNA probe (2 pM/μl) was denatured at 80°C for 5 min and then hybridized over night at 55°C. The slides were then washed in 0.2× SSC for 5 min at 57°C and then incubated in blocking buffer for 1 h at room temperature, and then in sheep Anti‐DIG Fab fragments, POD conjugated (Roche, Strasbourg, France) at room temperature overnight. Subsequently, the sections were managed with TSA(TM) Biotin System (PerkinElmer) for 30 min followed by cy3‐avidin (CST) for 2 h. 4′, 6‐Diamidino‐2‐phenylindole (DAPI) staining was used as the final step in the fluorescent staining procedure to label the cell nuclei. The semi‐quantitative analysis method of the ISH results and immunohistochemical analysis method is the same.