【Product Information】

【Product Description】

The circRNA hybridization kit includes a combination probe, a pre amplification structure, and an amplification probe. When two probes bind to adjacent positions of the target sequence, a small segment of the top sequence can bind to the pre amplification structure. The repeated sequence on the pre amplification structure can trigger a branched HCR to form a large nucleic acid aggregate. Each nucleic acid strand is labeled with digoxin, and the antibody recognizes digoxin for color development.

【Tissue Fixation】

【Storage and Shipping】

Ship on wet ice; store at -20℃ for long-term or at 4℃ for short-term use. Shelf life: 6 months.

【Protocol】

1. Deparaffinization and Rehydration

Immerse slides sequentially in:Xylene I (15 min) → Xylene II (15 min) → Xylene III (15 min) → 100% ethanol (10 min) → 90% ethanol (10 min) → 80% ethanol (10 min) → 70% ethanol (10 min) → Rinse with distilled water.

2. Repair

Cell Samples:After the sections are completely dry, use a histology pen (recommended: HKR14P in situ hybridization-specific histology pen) to draw a hydrophobic circle of appropriate size. Place the slides horizontally in an in situ hybridization instrument or a humid chamber. Apply 100 μL of Proteinase K repair solution (1X) onto the tissue and incubate at 37°C for 10 minutes. Rinse with pure water to stop the reaction.

Frozen Sections:For cell samples, after the sections are completely dry, use a histology pen (recommended: HKR14P in situ hybridization-specific histology pen) to draw a hydrophobic circle of appropriate size. Place the slides horizontally in an in situ hybridization instrument or a humid chamber. Apply 100 μL of Proteinase K repair solution (1X) onto the tissue and incubate at 37°C for 20 minutes (for frozen sections). Rinse with pure water to stop the reaction.

Paraffin Sections:Place the slide rack containing the slides into the repair chamber and pour in 1X citrate buffer (pH 6.0) repair solution (ensure the sample area is fully submerged). Cover the chamber, seal it with tape, and place it in a microwave oven. Heat at medium power for 8 minutes, rest for 8 minutes, then heat at medium-low power for another 8 minutes. Allow the solution to cool naturally, then block with 3% hydrogen peroxide for 15 minutes.

3. Blocking

Remove excess liquid from slides. Add 100 μL of Blocking Buffer per slide and incubate at 37℃ for 30 min. Wash once for 5 min.Wash steps: Place slide racks in a wash tank, add wash buffer (ensure samples are submerged), and shake at 60 rpm for 5 min.

4. Probe Hybridization

Remove excess liquid from slides. Add 100 μL of probe per slide and incubate at 37℃ for 3 h or overnight (maintain humidity to prevent drying). Wash 5 times, 5 min each.Wash steps: As described above.

5. Pre-Amplification Hybridization

Remove excess liquid from slides. Add 100 μL of Pre-Amplification Probe to each slide and incubate at 37℃ for 3 h or overnight (ensure humidity). Wash 5 times, 5 min each.Washing steps: As described above.

6. Probe Amplification

Remove excess liquid from slides. Add 100 μL of Amplification Probe per slide and incubate at 37℃ for 1.5 h (maintain humidity). Wash 5 times, 5 min each.Wash steps: As described above.

7. AP-Mouse Anti-Digoxin

Remove excess liquid from slides. Add 100 μL of AP-Mouse Anti-Digoxin (1X) per slide and incubate at 37℃ in a humidified chamber for 40 min. Wash 5 times, 5 min each.Wash steps: As described above.

8. Chromogenic Reaction

Prepare the chromogenic solution by adding 50 μL of NBT Chromogen Solution A and 50 μL of NBT Chromogen Solution B to 1 mL of deionized water. Add 100 μL of the mixture per slide and incubate at RT for 30 min. Rinse with distilled water to stop the reaction. Air-dry slides and mount with mounting medium.

【Precautions】

1.Wear lab coats and disposable gloves for safety.

2.For research use only.

【說明書下載】