Adenovirus DNA Probe to RNA1
This hyperbiotinulated DNA probe detects a very highly abundant, non-coding, early RNA species produced by all adenoviruses. The probe produces mostly nuclear staining in adenovirus infected cells and works well in formalin fixed tissues.
Preparation of the Probe Diluent:The probe is supplied in dry (lyophilized) form with one bottle of Probe Diluent.
To prepare the working Probe solution, transfer 1 ml of the Probe Diluent into the plastic vial containg the probe. Secure the cap on the vial and mix the solution by shaking it until the colored pellet is completely dissolved.
Transfer the new colored liquid into the original plastic bottle containing the rest of the Probe Diluent. Mix and write the probe description on the empty space provided on the label. The diluted probe is then ready to use. Keep the unused portion at -20 degrees C. AVOID REPEATED FREEZING AND THAWING CYCLES. DO NOT USE BEYOND THE EXPIRATION DATE INDICATED ON THE LABEL.
1. Heat tissue slides (FFPE) at 60C, 10 mins. dewax in xylene or appropriate dewaxing agent, rehydrate using 100%, 70%, 30% alcohol, buffer.
2. Incubate with Pepsin or proteinase K, for 20 mins.
3. Wash with probe wash 5X
4. wash with 95% alcohol 2X
5. wash with 100% alcohol 2X
6. Add 2-4 drops of Biotinylated probe provided, 10 mins. 100C
7. Let slide cool down to RT 3 mins
8. start hybridization 37C 30 mins.
9. Wash with Probe wash 5X.
10. Add 2-4 drops Avidin~Alk Phos. 37C 20 mins.
11. Wash with Probe wash 5X.
12. Add chromogen/substrate, 37C 15-30 mins.
13. Wash with distilled water 4-5 X
14. Counterstain, mount with Situ/Mount.
Freeze at -20 °C. ALIQUOT Repeated freezing and thawing should be avoided.