Preparation of cell samples with relatively uniform distribution of Alexa-647

1. Cells were grown on coverslips to around 80% confluence and subjected to the following assay, which was carried out at room temperature (around 22^oC) except specially indicating.
2. Cells were fixed with ice-cold 4% formaldehyde/PBS (freshly prepared from paraformaldehyde) for 10 min, followed by soaking in PBS for 5 min and rinsing once with ddH₂O.
3. Treat the fixed samples with freshly prepared sodium borohydride (1mg/mL) for 7 min. Make sure that bubbles were observed during sodium borohydride treatment. Soak samples in ddH₂O for 5 min, then remove the liquid.
4. Incubate samples with 0.5% Triton-X-100/PBS for 10 min, followed by rinsing with PBS for three times.
5. Prepare 5’-TTTTTTTTTT-3’ (10T) or Alexa-647-conjugated 10T (a 10T oligonucleotide labeled with one Alexa-647 at its 5’ end) at the indicated concentration in ddH₂O. Around 65µL solution was used for each coverslip in the following incubation steps.
6. To obtain samples with sparse or extremely sparse Alexa-647 density, cells on each coverslip were incubated overnight at 4˚C with
   65 mL 200 pM or 80 pM of Alexa-647-conjugated 10T, respectively. In the next day, samples were balanced at room temperature for around 20 min before washing with 2x SSC by 5 minutes for three times. To obtain samples with relatively high Alexa-647 density, samples were incubated with 2 nM of Alexa-647-conjugated 10T for 2 hr at room temperature, followed by rinsing with 2 x SSC for three times to remove free dye.
7. Fix cells on coverslips with freshly prepared ice-cold 4% formaldehyde/PBS for 10 min, followed by soaking in PBS for 5 min. If possible, incubate the fixed cells again in PBS for 40 min to remove formaldehyde.
8. Keep the samples at 4˚C for imaging.

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