Author: Stewart Laboratory ### FIX AND PROTEIN STRAIN 1. 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 1. 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 1. 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