Sheep anti dig alkaline phosphatase antibody

Four micrometer sections of human and mouse spinal cord and DRG were incubated with 50 µg/mL proteinase K, refixed in 10% formalin and treated with 0.5% acetic anhydride. RNA probes for human KIAA0319 and for its mouse homolog D130043K22Rik (NM_001081051) (both antisense and control sense probes) were synthesized and labeled with digoxigenin using the DIG RNA Labeling Kit (SP6/T7) (Roche). After incubating sections with prehybridization solution (50% formamide, 4×  SSC) for 2 h at 37°C, denatured probes (0.5 µg/µL) were mixed in hybridization buffer (40% formamide, 4× SSC, 1 mg/mL denatured sheared salmon sperm DNA, 1 mg/mL yeast t-RNA, 10 mM DTT, 10% dextran sulfate, 1× Denhardt's reagent) overnight. The stringency of washes varied according to the probe used. Following incubation for 2 h (37°C) with sheep anti-DIG-alkaline phosphatase antibody (1:1000, Roche), sections were developed overnight using NBT/BCIP (Roche). Photos were taken using an Olympus DP 25 microscope.

ISH was performed on paraffin-embedded sections of normal skin tissue obtained from Zyagen (San Diego, CA, USA) and psoriasis skin tissue obtained from Creative Bioarray (Shirley, NY, USA). Skin tissue slides were incubated with locked nucleic acid (LNA) probes (5′-digoxin [DIG]- and 3′-DIG-labeled LNA probes specific for miR-944 or scrambled probes with no homology to known vertebrate miRNAs; Exiqon, Woburn, MA, USA) overnight in a hybridization chamber at 40°C. After washing, probe binding was detected by incubating the slides with a sheep anti-DIG-alkaline phosphatase antibody (Roche, Mannheim, Germany). Slides were visualized after incubation in nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate solution (Thermo Scientific, Rockford, IL, USA).

Tissue microarrays containing 295 DGC and 112 IGC samples were used for in situ miRNA hybridization. In situ hybridization was performed using a miRCURY LNA miRNA ISH kit (Qiagen) with miR-199a-5p (5′-ACAGGTAGTCTGAACACT-3′) and miR-199b-5p (5′-AACAGATAGTCTAAACACT-3′) probes. The U6 and scramble miRNA probes provided in the kit were used as positive and negative controls, respectively. All probes were labeled with double digoxigenin (DIG) and denatured at 90 °C for 4 min before use. After deparaffinization and hydration, the slides were incubated with proteinase K (20 μg/mL) at 37 °C for 5 min (miR-199a-5p) or 15 min (miR-199b-5p). Slides were hybridized with probes against miR-199a-5p (50 nM, at 50 °C) and miR-199b-5p (125 nM at 40 °C) overnight in a hybridizer (ThermoBrite, Leica Biosystems, Richmond, IL). For probe detection, slides were incubated with sheep anti-DIG-alkaline phosphatase antibody (1:100; Roche, Mannheim, Germany) at room temperature for 1 h. The remaining steps were performed in accordance with the manufacturer’s instructions. The intensity of miR-199a and miR-199b expression was graded as follows: 0, no staining; 1, mild tumor cell staining; 2, moderate tumor cell staining; and 3, strong tumor cell staining. miR-199a and miR-199b expression levels were classified as low (0 and 1) and high (2 and 3), respectively.