FIX AND PROTEIN STRAIN:

1. Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
2. Wash cells with PBS +0.2% Tween20
3. Permeabilize cells with 0.5% Triton X-100 for 10’ at room temp.
4. Wash cells with PBS +0.2% Tween20
5. Block cells with blocking buffer for 15’ at 37^{°C (I use o.2% Tween20 and 10% serum)}
6. Stain cells with antibody of interest diluted in the blocking buffer for 60’ at 37°C. (I usually use dilution ranging from 1:100 to 1:500).
7. Wash 2 x 5’ in PBS + 0.2% Tween20.
8. Secondary antibody stain. Stain in blocking buffer for 45 to 60’ at 37°C.
9. Wash 2 x 5’ in PBS + 0.2% Tween20.

DNA FISH:

1. Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp.
2. Wash cells with PBS +0.2% Tween20
3. Wash 1 x in 2XSSC for 5’ at room temp.
4. Treat cells with RNAaseA diluted 1:100 (stock is 10 mg/ml) in 2X SSC for 45’ at 37°C.
5. Wash 1 x in 2XSSC for 5’ at room temp.
6. Dehydrate the slides:
   • 2’ in 70% EtOH
   • 2’ in 80% EtOH
   • 2’ in 100% EtOH
7. Air dry the slides
8. Set up hybridization mix:
   • Make blocking buffer (Roche 1096176, must be made up in 0.1M maleic acid and 150 mM NaCl, raise temp to 50-60°C and stir to get into solution).
   • Set up hyb: 70% Formamide
     • 0.3 μg/ml probe (if you use my aliquots you should add 5 mls to the tubes to get this concentration)
     • 1% blocking buffer
     • 10 mM Tris pH 7.2 (I often use 7.0)
9. Incubate at room temp for 2 hours in the dark.
10. Wash slides 2 x 15’ at room temp in 70% formamide + 10mM Tris pH 7.2
11. Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
12. Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20 + 0.1 ug.ml DAPI.
13. Wash slides 1 x 5’ at room temp in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20.
14. Dehydrate the slides:
    • 3’ in 70% EtOH
    • 3’ in 80% EtOH
    • 3’ in 100% EtOH
15. Air dry
16. Add mounting media and cover.