Immunofluorescence-Based Visualization of DNA Repair Protein Interactions

Abstract

Source: de la Peña Avalos, B., et al., Visualization of DNA Repair Proteins Interaction by Immunofluorescence. J. Vis. Exp. (2020).

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 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). 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. Prepare 4% (v/v) paraformaldehyde (PFA): dissolve 10 mL of 16% PFA aqueous solution in 30 mL PBS. Mi…

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

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.