Nbt bcip kit

In situ hybridizations were performed in 8-μm cryosections from the lung of mice with intra-bronchial administration Abs to H-2K^b or C1.18.4 isotype. In situ hybridizations of tissue sections were performed as previously described (17 (link)). After protease digestion, the digoxin-labeled locked nucleic acid (LNA)-scrambled control probe and LNA miR-16 and miR-195 antisense probe (Exiqon, Woburn, MA) were hybridized to the slides at 55°C for overnight. Following post hybridization washes with 0.2x SSC buffer at 55°C, 100 μl of rabbit anti-digoxin Ab (Sigma-Aldrich, Saint Louis, MO), diluted 1/500 in 10% sheep serum PBS buffer, was applied to the slides for 60 min at room temperature. Slides were counterstained with NBT-BCIP kit (Roche Lifescience, Indianapolis, IN), cover slipped, and mounted for viewing.

Chicken embryo mRNA distribution was determined by section in-situ hybridization, by use of a procedure described elsewhere [7 (link)].
After digoxigenin labeling (Roche, Indianapolis, IN, USA), the RNA probe was detected by either the chromogenic method or by use of fluorescent dye. For the chromogenic method, the probe was first recognized by use of anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) and then detected by use of the NBT/BCIP kit (Roche), in accordance with the manufacturer’s instructions. For fluorescence staining, the probe was first recognized by use of anti-digoxigenin antibody conjugated with peroxidase (Roche). The fluorescence signal was developed by use of the tyramide signal amplification kit (Perkin–Elmer, Waltham, MA, USA). The sequences of oligonucleotides used for probe production are listed in Supplementary Table 1.