Appendix 3 Ricardo V. Lloyd and Arie Perry

Albumin and Chromogranin ISH Microwave Method Day 1 (RNase free) Melt excess paraffin from slides in 70°C oven 15 min 1. Xylene x 2 7 min 2. 100% ETOH x 2 2 min 3. 95% ETOH x 2 2 min 4. 2 x SSC x 2 3 min 5. 0.2 N HCl (Pour fresh sol. from stock at R.T.) 20 min 6. 2 x SSC x 2 3 min Microwave 7. 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 heat sink of 400 ml H2O to control reaction. 10 min 8. Allow solution to cool 15 min 9. 2 x SSC 3 min Tissue digestion 10. Prot. K (25 ug/ml PBS) Stock prot K (500 mg/ml) is stored at -20°C. Thaw and dilute 1:20 with PBS (conc = 25 mg/ml). Wipe around tissue and cover with 200-300 µl. Incubate in humid tray in 50° oven. 10 min 11. 2 x SSC x 3 3 min 12. 0.1 M Triethanolamine 200 ml. Add 1.2 ml Acetic Anhydride immediately after adding slides. 15 min 13. 2 x SSC 5 min Prehybridization, Denaturation, and Hybridization 14. Prehybridization buffer at R.T. Wipe around tissue and cover with 200-300 µl. Incubate in humid tray. 1 hour 15. Hybridization - Drain off the prehybridization buffer, wipe around tissue, add appropriate probe, coverslip and incubate in humid tray in 50° oven. overnight Day 2 (Not RNase free) Stringent Wash 1. Wash in 2 x SSC (preheated to 37 C*) 10 min 2. Wash in 1 x SSC (preheated to 37 C*) #2 setting on 37 C Shaking 10 min 3. Wash in 0.5 x SSc (preheated to 37 C*) Waterbath 10 min 4. 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 5. Incubate in humid tray at R.T. Buffer A with 1% normal sheep (or swine) serum, 0.3% Triton X-100. Wipe around tissue before adding enough to cover each slide. (If doing many slides, use a coplin jar). 30 min 6. Incubate in humid tray in 370C oven. Make a 1:200 dilution of Anti- digoxigenin-AP (stored at 4°C), using Buffer A 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. 2 hours 7. Buffer A x 2 on Orbital Shaker 3 min 8. Buffer C on Orbital Shaker 3 min 9. Incubate in humid tray at R.T. (wrapped in foil). NBT/BCIP with Levamisole: 8 µl 125nM Levamisole (stored at 4°C) 4.4 µl NBT (stored at -20°C) 3..3 µl BCIP (stored at -20°C) 1 ml Buffer C Make enough to cover all slides after wiping around each tissue. 1.0 hours 10. Pour off reagent and put in Buffer C 5 min 11. Pipette Nuclear Fast Red on each tissue 3 min 12. Rinse slides in Buffer C 1 min 13. Dehydrate and coverslip with Permount.