【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. Enzyme Repair

After slides are completely dry, draw a hydrophobic circle around the tissue using a histology pen (recommended: HKR14P In Situ Hybridization Pen). Place slides horizontally in a hybridization oven or humidified chamber. Add 100 μL of Proteinase K repair solution (1X) onto the tissue and incubate at 37℃ for 30 min (15 min for cell samples). Rinse with distilled water to stop the reaction.

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.Wash 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. HRP-Mouse Anti-Digoxin

Remove excess liquid from slides. Add 100 μL of HRP-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

Remove excess liquid from slides. Add 100 μL of TSA Chromogenic Solution per slide and incubate at RT for 10 min. Rinse with distilled water to stop the reaction.

9. DAPI Staining

Add 50 μL of DAPI staining solution per slide, incubate in the dark for 5 min, rinse with distilled water, and mount with anti-fade mounting medium.

【Precautions】

1.Wear lab coats and disposable gloves for safety.

2.For research use only.

【說明書下載】