Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Overview

In this video, we describe the immunofluorescence method to visualize the localization of the DNA repair proteins γH2AX and 53BP1 at the sites of double-strand DNA breaks in irradiated cell nuclei.

Protocol

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.

Antibody	Company	Reference	Source
53BP1	Cell Signaling	4937	Rabbit
Anti-Mouse IgG H&L (Alexa Fluor 647)	Abcam	ab150103	Donkey
Anti-phospho-Histone H2A.X (Ser139), clone JBW301	Millipore	05-636	Mouse
Anti-Rabbit IgG H&L (Alexa Fluor 488)	Abcam	ab150081	Goat

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.

Representative Results

Figure 1. Nuclear extraction.

Representative images of cells prior to (left) and post (right) nuclear extraction. Cytoplasm should be digested but the nuclear structure should remain intact post-extraction (right). (A) 20x magnification; scale bar = 20 μm and (B) 40x magnification; scale bar = 10 μm.

Materials

Name	Company	Catalog Number	Comments
Coverglass #1, 18 mm round (coverslips)	Neuvitro	NC0308920	Coverslips need to be cleaned and sterilized prior using, with HCl or ethanol.
DPBS, no calcium, no magnesium	Gibco	14190144	PBS for tissue culture
SlowFade Diamond Antifade Mountant with DAPI	Invitrogen	S36973	300 nM DAPI with VECTASHIELD Antifade Mounting Medium can be used instead.
Bovine serum albumin (BSA)	Sigma-Aldrich	A3059	Heat-shock fraction is recommended, to avoid precipitation/background.
Triton X-100	AmericanBio	AB02025	Nuclear extraction buffer.
Superfrost Plus Microscope Slides	Fisherbrand	1255015	Polysine Slides can be used instead.