Lna modified and dig labeled oligonucleotide

ISH was conducted on paraffin embedding samples of NSCLC to investigate the clinical response to TKI and miR-21 expression. Briefly, slides were treated and hybridized with 10 pmol probe (LNA-modified and DIG labeled oligonucleotide; Exiqon) complementary to miR-21, according to the manufacturer’s instructions. After incubation with anti-DIG-HRP fab fragments conjugated to horseradish peroxidase, the hybridized probes were detected by incubation with 3′3-diaminobenzidine solution with nuclei counterstained with Carazzi’s haematoxylin. Staining patterns were analyzed by 3 experts and the proportion of positively stained tumor cells was graded as follows: 0 (no positive cells), 1 (<10% positive cells), 2 (10%–50% positive cells), 3 (>50%positive cells). Cells at each intensity of staining were recorded on a scale of 0 (no staining), 1(weak staining, light blue or yellow), 2 (moderate staining, blue or yellow), and 3 (strong staining, dark blue or yellow). For tumors that showed heterogeneous staining, the predominant pattern was considered for scoring. The staining index (SI) was calculated as proportion of positively stained tumor cells×staining intensity. Using this method, the expression of miR-21 was scored as 0, 1, 2, 3, 4, 6, or 9. In case of disagreement (score discrepancy>1), slides were reexamined and a consensus was reached by the experts.

Tissue microarrays were stained with primary antibodies against HK1 (Cat. no. sc-46695; Santa Cruz), SLC2A1 (Cat. no. sc-377228; Santa Cruz), RUNX1 (Cat. no. ab23980; Abcam). The in situ detection of miR-30d was performed on 6-μm formalin-fixed, paraffin-embedded (FFPE) sections using DIG-labeled miRCURYTM Detection probe (Exiqon, Denmark). Briefly, the slides were hybridized with a probe (LNA-modified and DIG-labeled oligonucleotide; Exiqon) complementary to miR-30d and after incubation with anti–DIG-AP Fab fragments conjugated to alkaline phosphatase. The hybridized probes were then detected by applying nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color substrate (Roche) to the slides. Slides were counterstained with VECTOR® nuclear fast red counterstain (VECTOR LABOTATORIES) and analyzed with a Nikon 80i microscope and Nikon NIS-Elements F 2.3 software (Nikon).