Unless noted otherwise, all incubations take place at room temperature

1. Equilibrate frozen sections to RT
2. Wash in 2xSSC, incubate two times, 3 min each incubation (2x3min)
3. Incubate in 0.2 M HCL for 10 min at room temperature
4. Wash in 2xSSC 2x3min
5. Incubate in 1M NaSCN for 20 min at 75-80°C
6. Wash in 2xSSC 2x3min
7. Water rinse
8. Ethanol dehydrate (70%, 95%, 100%), 1 min each
9. Air-dry slides
10. Hybridization: pre-heat probe to 75°C, vortex to mix
    a. Add 10-12 µl 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
    
11. Carefully remove rubber cement and coverslip, wash in 2xSSC 2 min
12. Wash with 0.4x SSC/0.3%NP40 at 55 C for 3 min.
13. Wash 2xSSC 2 min
14. Block: incubate in 4xSSC/3%BSA/0.1%Tween20 for 30 min at 37°C
15. 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.
16. Wash well with 2xSSC
17. Rinse slides in water
18. Air-dry slides in the dark
19. 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