4 nitroblue tetrazolium chloride nbt

Digoxigenin (DIG)-labeled sense and antisense cRNA probes corresponding to the coding region of mouse TNFR1 and TNFR2 were synthesized using the DIG RNA labeling kit (Roche Applied Science). Fresh-frozen taste sections (10 µm/section) from C57BL/6J mice (4-6 months old) were attached to clean glass slides. Sections were then fixed with 4% paraformaldehyde and processed for in situ hybridization as previously described (Wang et al., 2007 (link)). Hybridizations were performed at 72°C overnight with DIG-labeled probes in 50% formamide, 5× SSC, 5× Denhardt's solution, 250 µg/ml yeast RNA, and 500 µg/ml sperm DNA. Sections were washed three times at 72°C with 0.2× SSC. Hybridized DIG-labeled cRNA was detected immunologically with an alkaline-phosphatase-conjugated anti-DIG antibody and standard chromogenic substrates 4-Nitro Blue tetrazolium chloride (NBT, Roche Applied Science). Images were taken using a Nikon fluorescence microscope. In all the experiments, hybridizations to antisense and sense probes were performed in parallel to verify the specificity of hybridization signals.