| A. De-paraffinization(RNasefree) |
| Melt excess paraffin from slides in 70° oven | 15 min |
| Xylene x 2 | 7 min |
| 100% ETOH x 2 | 2 min |
| 95% ETOH x 2 | 2 min |
| DEPC H2O | 3 min |
| Weigert's Iodine; rinse slides with DEPC H2O | 5 min |
| 5% sodium thiosulfate | 1 min |
| DEPC H2O | 3 min |
| B. Tissue Digestion (RNase free) |
| Proteinase K: 25 ug/ml PBS (1:20 dil. of stock Prot. K). Incubate in humid tray in 50° incubator. | 15 min |
| DEPC H2O x 2 | 5 min |
| 0.1 M Triethanolamine 200 ml. Add 1.2 ml Acetic Anhydride immediately after adding slides to the Triethanolamine. | 15 min |
| DEPC H2O | 5 min |
| C. Denaturation & Hybridization (RNase free) Probe |
| Cover tissue sections with prehybridization buffer. | 5 min |
| Drain prehyb. buffer from slidea, wipe excess from around tissue,and add approx.20 ml 0.1 ng/ l PAAV-2 to negative control slides. Add approximately 20 ml of Probe to test slides, add sigmacoted coverslips to all slides, place them on metal tray in 100° oven | 5 min |
| Incubate slides in humid tray in 50° oven | 2 hr |
| D. Stringent Wash |
| Place slides in 2xSSC (rotate) | 5 min |
| Place slides in 1xSSC | 2 min |
| Wash slides in 1xSSC heated to 50°. | 30 min |
| Place slides in 1xSSC | 1 min |
| E. Detection |
| Wipe around tissue, add 1-2 drops (20-40 ml) anti-fluorescein-AP (Bottle #3) put in humid tray at R.T. | 20 min |
| 1xSSC on Orbital Shaker | 3 min |
| Buffer C on Orbital Shaker | 3 min (for Buffer C) |
| Incubate in humid tray at R.T. (wrapped in foil). NBT/BCIP with (Bottle #4): Make enough to cover all slides after wiping around each tissue. | 1 hr |
| Pour off reagent and put in Buffer C | 5 min |
| F. Counterstain and Coverslip |
| Pipette Nuclear Fast Red on each tissue | 1 min |
| Rinse slides in Buffer C | 1 min |
| 95% ETOH x 2 | 2 min min |
| 100% ETOH x 2 | 2 min |
| Xylene x 2 | 7 min |
