ISH Protocol with Photosensitive Biotin Nucleic Acid Probes

Photosensitive biotin has a linker arm with biotin attached at one end and an aryl azide compound at the other. Under visible light irradiation, the aryl azide compound may become activated aryl nitrobenzene, which readily binds specifically to the adenine N-7 position of DNA or RNA, binding approximately one biotin per 50 bases. We present a method for in situ hybridization of chromosomes with a photosensitive biotin-labeled nucleic acid probe.

• Paraffin sections are dewaxed into the water and rinsed in 0.1 mol/L PBS (pH 7.2) for 5 min; frozen sections are rinsed directly into PBS for 5 min.
• Rinse with 0.1 mol/L glycine PBS for 5 min, then rinse with 0.4% Trition X-100 PBS for 15 min.
• Hold in proteinase K 1 μg/ml (0.1 mol/L Tris-HCl pH 8.0, 50 mmol/L EDTA) at 37°C for 30 min.
• Fix in 4% paraformaldehyde PBS for 5 min.
• Rinse with 0.1 mol/L PBS for 2×3 min.
• Place in 0.25% acetic anhydride (0.1 mol/L triethanolamine) for 10 min.
• Rinse with 2×SSC for 10 min (1×SSC including 0.15 mol/L NaCl, 0.015 mol/L sodium citrate).
• Take 10 μl drops of hybridization solution containing the corresponding probe on the specimen. If it is a cDNA probe then hold the probe in a 95°C water bath for 10 min before using it, immediately put it into an ice bath to cool down, and then use it again.
• Cover with a 22×22 mm siliconized cover sheet or a suitable size of wax film and put into a warm box at 43°C for 12-16h.
• Elute the cover sheet with 4×SSC and rinse in the same solution at 37°C for 10-30 min.
• Rinse with 2×SSC (containing 20 μg/ml RNaseA, suitable for RNA probes) for 30 min at 37°C.
• Rinse with 1×SSC, 0.1×SSC, 37°C for 10-30 min each.
• Wash with 0.05 mol/L PBS for 4×5 min.
• Hold in 3% BSA (0.4% Triton X-100 PBS with) for 30 min at 37°C.
• Place in Avidin-AKP (alkaline phosphatase) (1:500-1:100, 0.4% Triton X-100 PB) at room temperature for 1-3 h.
• Rinse with 0.05 mol/L PBS for 4×5 min; TSM1 for 2×5 min; TSM2 for 2×5 min.
• Mix 0.4 mg/ml nitro tetrazolium blue (NBT) and 0.2 mg/ml 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for color development, at room temperature in a dark place for 3 h. Terminate the color development with 20 mmol/L EDTA (pH 8.0).
• Glycerol gelatin is used to seal the film directly.

Creative Bioarray Relevant Recommendations

• A labeled RNA or DNA probe can be used to hybridize to a known target mRNA or DNA sequence within a sample. Creative Bioarray offers different types of ISH Probes and Custom Probes. Tell us the specific gene or the chromosome region of your interest, and the desired dye color, and we will make a custom probe for you.

• In addition to a certain concentration of labeled probes, the hybridization solution contains higher concentrations of salts, formamide, dextran sulfate, bovine serum albumin, and carrier DNA or RNA.
• A higher concentration of Na⁺ in the hybridization solution increases the hybridization rate and can reduce electrostatic binding between the probe and the tissue specimen.

Probe

In Situ Hybridization (ISH) & RNAscope Service

Digital ISH Image Quantification and Statistical Analysis