Author: Stewart Laboratory
FIX AND PROTEIN STRAIN
- Fix cells in 4% paraformaldhyde (diluted in PBS) for 10min at RT
- Wash cells with PBS +0.2% Tween20
- Permeabilize cells with 0.5% Triton X-100 for 10 min at RT
- Wash cells with PBS +0.2% Tween20
- Block cells with blocking buffer for 15 min at 37°C (We use 0.2% Tween20 and 10% serum)
- Stain cells with antibody of interest diluted in the blocking buffer for 60 min at 37°C. (We usually use dilutions ranging from 1:100 to 1:500)
- Wash 2x 5 min in PBS + 0.2% Tween20
- Secondary antibody stain. Stain in blocking buffer for 45 to 60 min at 37°C
- Wash 2x 5 min in PBS + 0.2% Tween20.
DNA FISH
- Fix cells in 4% paraformaldhyde (diluted in PBS) for 10’ at room temp
- Wash cells with PBS +0.2% Tween20
- Wash 1 x in 2XSSC for 5’ at room temp
- Treat cells with RNaseA diluted 1:100 (stock is 10 mg/ml) in 2X SSC for 45’ at 37°C
- Wash 1 x in 2XSSC for 5’ at room temp
- Dehydrate the slides:
- 2 min - 70% EtOH
- 2 min - 80% EtOH
- 2 min - 100% EtOH
- Air dry the slides
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 our aliquots you should add 5 mL to the tubes to get this concentration)
- 1% blocking buffer
- 10 mM Tris pH 7.2 (We often use 7.0)
- Incubate for 2 hrs at RT in the dark
- Wash slides 2x 15 min at RT in 70% formamide + 10mM Tris pH 7.2
- Wash slides 1x 5 min at RT in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20
- Wash slides 1x 5 min at RT in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20 + 0.1 ug.mL DAPI
- Wash slides 1x 5 min at RT in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20
- Dehydrate the slides:
- 3 min - 70% EtOH
- 3 min - 80% EtOH
- 3 min - 100% EtOH
- Air dry
- Add mounting media and cover
