ISH Protocol for Determination of TNF-mRNA

Tumor Necrosis Factor (TNF) induces apoptosis in a variety of cancer cells playing a role in cancer growth and metastasis. In situ hybridization (ISH) is a technique that uses colorimetric or fluorescent probes to target and visualize specific DNA or RNA sequences within tissues. Here, we present the mRNA of TNF determined by in situ hybridization.

In situ hybridization

• Prepare hybridization solution, including 60% deionized formamide, 300 mmol/L NaCl, 30 mmol/L sodium citrate, 10 mmol/L EDTA 25 mmol/L NaH₂PO₄ (pH 7.4), 5% dextran sulfate, 250 μg/μl denatured salmon sperm DNA.
• Prepare digoxigenin-labeled DNA probe, before use, heat the DIG-labeled DNA probe at 80°C for 10 min, rapidly denature it in an ice bath, and add it to the hybridization solution to a final concentration of 5 μg/μl.
• Add 10-20 μl of hybridization mixture (hybridization solution plus denatured probe) to the fixed and increased permeability cells, cover with a special in situ hybridization coverslip, and put it into a wet box containing about 20 ml of 20% glycerol at 37°C to make it hybridize over the solution.

Washing

• Prepare washing solution. 2×SSC solution is in 1000 ml distilled water with 17.6 g sodium chloride and 8.8 g trisodium citrate (C₆H₅O₇NA₃-2H₂O, molecular weight 294), 0.5×SSC solution is in 300ml distilled water with 100 ml 2×SSC, 0.2×SSC is in 270 ml distilled water with 30 ml 2×SSC.
• Remove the coverslip and wash 2×SSC at about 30-37°C water temperature for 5 min×2 times, 0.5×SSC for 15 min×1 times and 0.2×SSC for 15 min×1 times.

Fluorescent antibody detection

• Prepare a blocking solution containing 100 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, and 0.5% sheep serum, prepare a washing solution, which is 0.1 M PBS (pH 7.2-7.4), prepare the antifade solution, nine parts glycerol with one part staining solution (1 mmol/L Tris-HCl (pH 7.5), 2% 1.4-diaza- bicyclo [2,2,2]-Octane, 500 ng/ml propidium iodide).
• Add 100 μl of blocking solution on each slide to block the non-specific binding site.
• Add anti-DIG-fluorescein antibody to the slide at 1:500 dilution with the blocking solution, place in a wet box at 37°C for 45 min, and wash with washing solution for 5 min×4 times.
• Wash the slides with 100 mol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.05% Tween 20.
• Soaking the cell samples in 70%, 90%, and 100% ethanol for 5 minutes in that order and dehydrating them.
• Dry the slides in the air, place the cell samples in the anti-fading solution, and remove the sealed slides.

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• All solutions, glassware, forceps, etc. must be RNase-free up to the hybridization step.
• Preheat the DIG-labeled DNA probe at 80°C for 10 min before use.

Probe

In Situ Hybridization (ISH) & RNAscope Service

Digital ISH Image Quantification and Statistical Analysis