STEWART LABORATORY

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PROTEIN-DNA TELOMERE FISH

 

FIX AND PROTEIN STRAIN

 

                                                      1.            Fix cells in 4% paraformaldhyde (diluted in PBS) for 10min at RT

 

                                                      2.            Wash cells with PBS +0.2% Tween20

 

                                                      3.            Permeabilize cells with 0.5% Triton X-100 for 10 min at RT

 

                                                      4.            Wash cells with PBS +0.2% Tween20

 

                                                      5.            Block cells with blocking buffer for 15 min at 37C (We use 0.2% Tween20 and 10% serum)

 

                                                      6.            Stain cells with antibody of interest diluted in the blocking buffer for 60 min at 37C. (We usually use dilutions ranging from 1:100 to 1:500)

 

                                                      7.            Wash 2x 5 min in PBS + 0.2% Tween20

 

                                                      8.            Secondary antibody stain. Stain in blocking buffer for 45 to 60 min at 37C

 

                                                      9.            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

 

                                                      2.            Wash cells with PBS +0.2% Tween20

 

                                                      3.            Wash 1 x in 2XSSC for 5 at room temp

 

                                                      4.            Treat cells with RNaseA diluted 1:100 (stock is 10 mg/ml) in 2X SSC for 45 at 37C

 

                                                      5.            Wash 1 x in 2XSSC for 5 at room temp

 

                                                      6.            Dehydrate the slides:

 

         2 min - 70% EtOH

 

         2 min - 80% EtOH

 

         2 min - 100% EtOH

 

                                                      7.            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 50-60C and stir to get into solution)

 

                                                      2.            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)

 

 

                                                      3.            Incubate for 2 hrs at RT in the dark

 

                                                      4.            Wash slides 2x 15 min at RT in 70% formamide + 10mM Tris pH 7.2

 

                                                      5.            Wash slides 1x 5 min at RT in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20

 

                                                      6.            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

 

                                                      7.            Wash slides 1x 5 min at RT in 0.05 M Tris pH 7.2 + 0.15 M NaCl + 0.05% Tween20

 

                                                      8.            Dehydrate the slides:

 

         3 min - 70% EtOH

 

         3 min - 80% EtOH

 

         3 min - 100% EtOH

 

                                                      9.            Air dry

 

                                                 10.            Add mounting media and cover