Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

1. Nuclear extraction and cell fixation

1. Prepare stock solutions: 0.2 M PIPES (pH 6.8), 5 M NaCl, 2 M sucrose, 1 M MgCl₂, 0.1 M EGTA (pH 8.0).
2. Prepare nuclear extraction buffer (NEB): dissolve 10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA (pH 8.0) and 0.5% (v/v) Triton X-100 in ddH₂O. Mix until dissolved completely.
3. Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mix until dissolved completely.
4. At appropriate time point (t=0, 1, 2, 4, 16 h), wash cells twice with 1 mL of PBS. Remove PBS completely.
5. Add 200 μL of NEB to each well for cell nuclei extraction (cytoplasm is degraded, only nucleus remains) (Figure 1). Incubate for 2 minutes at room temperature and remove completely.
   NOTE: Do not exceed 2 minutes.
6. Wash cells with 1 mL of PBS. Remove PBS completely. Add PBS carefully, cells are very fragile at this step.
7. Add 200 μL of 4% (v/v) PFA to each well for cell fixation. Incubate for 10 minutes at 4 °C. Remove PFA completely.
8. Add 1 mL of PBS.
   NOTE: Cells can be stored in PBS at 4 °C.

2. Immunofluorescence staining

1. Prepare blocking solution: dissolve 5% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved.
2. Prepare dilution buffer: dissolve 1% BSA (w/v) in PBS and add 0.3% (v/v) Triton X-100. Mix until completely dissolved.
3. For blocking, add 200 μL of blocking solution to each well. Incubate for 2 hours at room temperature or 16-18 hours at 4 °C.
   NOTE: If goat antibody will be used, add 5% goat serum to blocking solution.
4. Dilute primary antibody in dilution buffer (1:500; see Table 1 for antibodies list) and vortex until well mixed.
   NOTE: If goat antibody is used, add 1% goat serum to dilution buffer.
5. In a humidity/incubation box, adhere a piece of parafilm. Add 10 μL of primary antibody (in a single drop). Align one edge of the coverslip with the drop and slowly lower onto the parafilm, for the liquid to spread throughout (avoid bubbles if possible). Incubate for 2 hours at room temperature.
6. Wash coverslips three times in PBS for 1 minute.
7. Dilute secondary antibody in dilution buffer (final concentration: 2 μg/mL) and vortex until well mixed.
8. Apply 10 μL of secondary antibody as described for the primary antibodies. Incubate for 2 hours at room temperature.
   NOTE: Protect from light.

Table 1: Antibodies used. List of antibodies used in this study.

9. Wash coverslips three times in PBS for 1 minute.
10. Wash coverslips with H₂O for 1 minute.
11. Counterstain DNA with DAPI: apply 10 μL of 300 nM DAPI (as described for antibodies), incubate for 30 minutes at room temperature and then mount onto glass slide with a glycerol based mounting media. Alternatively, add one drop (10 μL) of commercial antifade mounting media containing DAPI onto a slide and apply a coverslip. Gently press the coverslip and remove excess fluid around it with a paper towel.
12. Seal coverslips with transparent nail polish and let them dry for 20 minutes.
13. Store slides at 4 °C.

3. Image acquisition

1. Place a drop of immersion oil onto the 60x objective lens. Use DAPI to locate the nuclei through eye piece.
   1. For XYZ image acquisition, open acquisition software and select parameters: Scanner type: Galvano; Scanner mode: Roundtrip; Image size: 512×512; PMT mode: VBF; PMT average: frame (4 times); PMT sequential scan: line.
   2. Select the dye and the detectors:
      Channel (CH1), Dye (DAPI), Detector (SD1)
      Channel (CH2), Dye (Alexa Fluor 488), Detector (HSD3)
      Channel (CH3), Dye (Alexa Fluor 647), Detector (HSD4)
   3. Select ON in "Z".
2. Adjust the live image. Press the Live button on the Live window.
   1. Adjust the focus and set laser intensity (%), sensitivity (HV), gain and offset on "PMT" tool window.
      1. Adjust the laser intensity (%): for brightness and bleaching. The higher the laser intensity, the stronger the signal, but the specimen will photobleach.
      2. Adjust the sensitivity (HV): noise level. The higher the HV, the stronger the signal, but image will be noisy if too high.
         NOTE: Always keep voltage constant.
      3. Adjust the offset: background level.
   2. Select Start/End (15 slices), for Z stacks.
3. Start the acquisition.
   1. Select the folder to save images. Press the LSM Start button to start acquiring the image. Press the Series Done button to complete the image acquisition.