Unless noted otherwise, all incubations take place at room temperature

1. Heat tissues to 60°C for 10 mins
2. Deparaffinize and rehydrate tissue sections through series of xylenes and ethanol incubations
   a. Xylenes or alternative (Citrisolve, Histoclear) incubate 2 times, 5 min each incubation (2 x 5 min)
   b. 100% ethanol, 5 min
   c. 95% ethanol, 5 min
   d. 70% ethanol, 5 min
   
3. Rinse the slides once in dH20
4. Wash with PBS for 5 mins
5. Antigen retrieval: Preheat BD retrievagen A solution (BD Pharmigen cat# 550524) in coplin jar in steamer. Add slides to coplin jar and incubate for 10-20 min in steamer then let stand at room temperature for 20 min.
6. Wash 2xSSC 2x3min
7. Incubate in 0.2 M HCL for 12 min at room temperature
8. Wash in 2xSSC 2x3min
9. Incubate in 1M NaSCN for 20 min at 75-80°C
10. Wash in 2xSSC 2x3min
11. Water rinse
12. Ethanol dehydrate (70%, 95%, 100%), 1 min each
13. Air-dry slides
14. Hybridization: pre-heat probe to 75°C, vortex to mix
    a. Add 10-12µl preannealed probe to slides and cover with a small coverslip (18mmx18mm).
    b. Seal with rubber cement - must be certain that edges of coverslip are sealed completely (can’t use too much!)
    c. Incubate @ 75°C for 5 min.
    d. Incubate @ 37°C overnight in moist chamber
    
15. Carefully remove rubber cement and coverslip, wash in 2xSSC 2 min
16. Wash with 0.4x SSC/0.3%NP40 at 55°C for 3 min.
17. Wash 2xSSC 2 min
18. Block: incubate in 4xSSC/3%BSA/0.1%Tween20 for 30 min at 37°C
19. Detection: dilute anti DIG-rhodamine (Roche cat# 1 207 750) 1:20 in 4xSSC/1%BSA/0.1%Tween20 and incubate at 37°C for 45 min.
20. Wash well with 2xSSC
21. Rinse slides in water
22. Air-dry slides in the dark
23. Add a drop of vectashield mounting medium with DAPI (Vector cat# H-1200), coverslip and view.

Solutions:

Blocking Solution

For 100 mls:
3g BSA - end conc. 3% BSA
20mls 20x SSC - end conc. 4x SSC
100µl Tween20 - 0.1% Tween in 100mls
H2O up to 100 mls

Detection Solution

For 100 mls:
1g BSA - end conc. 1% BSA
4xSSC/0.1% Tween up to 100 mls

Hybridization Mixture

For 20 mls:
8 mls 50% Dextran Sulfate - end conc. 20% Dextran Sulfate
4 mls 20x SSC - end conc. 4x SSC
8 mls H2O