| A. Deparaffinization (RNase free) |
| - Melt excess paraffin from slides in 70 C oven | 15 min |
| - Xylene x 2 | 7 min |
| - 100% ETOH x 2 | 2 min |
| - 95% ETOH x 2 | 2 min |
| - 2 x SSC x 2 | 2 min |
| - 0.2 N HCL | 20 min |
| - 2 x SSC x 2 | 3 min |
| - Weigert's Iodine; rinse slides with DEPC H2O | 5 min |
| - 5% sodium thiosulfate | 1 min |
| - 2 x SSC | 3 min |
| B. Microwave (RNase free) |
| - Use RNase free plastic coplin jars. Add slides to jar(s) filled with 10 mM Citrate pH 6.0. Microwave for 4-5 min increments - refill jars with DEPC H2O when solution levels drop. Use 400 ml H2O in beaker as a heat sink. | 10 min |
| - Allow solution to cool | 15 min |
| - 2 x SSC at R.T. | 3 min |
| C. Tissue Digestion (RNase free) |
| - Proteinase K: 25 g/ml PBS (1:20 dil. of stock Prot. K). Incubate in humid tray in 50 C° incubator | 5 min |
| - 2 x SSC x 2 | 3 min |
| - 0.1 M Triethanolamine 200 ml. Add 1.2 ml Acetic Anhydride immediately after adding slides. | 15min |
| - 2 x SSC | 5 min |
| - Hybridization: Add JCV probe (1 ng/ul) to ISH slide and PAAV-2 (0..1 ng/ul) to patient negativecoverslip, place them on metal tray on 100 oven. | 5 min |
| - Incubate slides in humid tray in 50 C oven | 2 hr |
| D. Stringent Wash |
| - Wash in 2 x SSC (preheated to 37 C*) | 10 min |
| - Wash in 1 x SSC (preheated to 37 C*) #2 setting on 37 C Shaking | 10 min |
| - Wash in 0.5 x SSc (preheated to 37 C*) Waterbath | 10 min |
| - Buffer A~ * Preheat to 42 C when using staining jars on the orbital shaker. The temperature of solution will decrease to the optimum 37 C once it is poured into the staining jar. | 1 min |
| E. Detection |
| - Incubate in humid tray in 37° oven. Make a 1:200 dilution of Anti- digoxigenin-AP (stored at 4°), using the Buffer with 1% normal sheep (or swine) serum, 0.3% Triton X-100. Drain slides, wipe around tissue and add enough to cover each section well. | 20 min |
| - Buffer A x 2 on Orbital Shaker | 3 min |
| - Buffer C | 3 min |
- Incubate in humid tray at R.T. (wrapped in foil). NBT/BCIP with Levamisole:
3.2μl BCIP (BRL) stored at -20°C
4.4μl NBT (BRL) stored at -20°
8.0μl Levamisole - stored at 4°C
1ml Buffer C
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 |
| - Dehydrate and coverslip with Permou | |
