Alkaline phosphatase conjugated second antibody

Tisue microarray samples for ovarian tumours were used for miR-192 quantification. The formalin-fixed paraffin-embedded tissue sections were dewaxed in xylenes, and rehydrated through an ethanol dilution series. The tissue sections were digested with 15 μg ml⁻¹ proteinase K for 20 min at room temperature, then loaded onto Ventana Discovery Ultra for in situ hybridization analysis16 (link). The tissue slides were incubated with double-DIG-labelled mercury LNA miR-192 probe (Exiqon) for 2 h at 52 °C. The double-DIG-labelled control U6 snRNA probe (Exiqon) was used as a positive control. The digoxigenins were then detected with a polyclonal anti-DIG antibody and alkaline phosphatase conjugated second antibody (Ventana) using NBT-BCIP as the substrate. Representative light field images were obtained using a Nikon Microphot-FXA microscope and Leica DFC320 digital camera. The expression levels of miR-192 were determined using CellProfiler 2.0 software to establish the staining intensity threshold levels and to quantify the number of positively stained cancer cells per HPF ( × 200 magnification)16 (link).

Frozen tissue sections were first digested with 5 μg/mL proteinase K for 5 min at room temperature and were then loaded onto Ventana Discovery Ultra system (Ventana Medical Systems, Inc, Tucson, AZ, USA) for in situ hybridization analysis. The tissue slides were incubated with double-DIG labeled custom LNA probe for PRKAR1B-AS (Exiqon, Qiagen, Hilden, Germany) for 2 h at 55 °C. The digoxigenins were detected with a polyclonal anti-DIG antibody and alkaline phosphatase–conjugated second antibody (Ventana, Oro Valley, AZ, USA) using NBT-BCIP as the substrate. The double-DIG labeled control U6 snRNA probe was also from Exiqon.

The 4-μm paraffin-embedded TMA sections were hybridized with the double-DIG-labelled miRCURY LNATM detection probe, hsa-miR-506 (38314-15, Exiqon, Woburn, MA, USA) for 2 h at 55°C, as described previously [13 (link), 14 (link)]. The digoxigenins were detected with a polyclonal anti-DIG antibody and an alkaline phosphatase-conjugated second antibody (Ventana, Tucson, AZ, USA), using Nitroblue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP) as the substrate. The LNA U6 snRNA probe (Exiqon, Woburn, MA, USA) was used as a positive control for every TMA core, with blue staining in the nucleus. For miR-506, blue staining in the cytoplasm was defined as positive signals. Signals in tumor cells and pancreatic non-tumor tissue (pancreatic ducts, acinar cells, and islands) were quantified by two senior pathologists, using a previously described scoring system [13 (link), 14 (link)] with some modifications. The signal intensity (0, no signal; 1, weak signal; 2, intermediate signal; and 3, strong signal) and the percentage of positive cells (0, 0%; 1, <25%; 2, 25%–50%; and 3, >50%) were multiplied to obtain a score (0, 1, 2, 3, 4, 6, or 9). Low and high miR-506 expression levels were defined as scores of <4 and ≥4, respectively.