Indirect immunofluorescence with preextraction (in situ cell fractionation)
Lab/Group: d'adda di fagagna's group
Related Journal & Article Information
Journal: Nature
Article Title: Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication
Introduction
Immunostaining of DDR proteins in senescent cells
The study of the activation of DNA damage response factors at the single cell level relies on the observation of their accumulation (or the accumulation of their activated forms) in nuclear foci by plain immunofluorescence techniques. Nevertheless, a number of them such as ATR, ATRIP and RPA do not form detectable foci if their excess is not first removed by a brief treatment that washes away the fraction that is not chromatin bound. This describes here this technique first reported by Mirzoeva, O. K. & Petrini, J. H. J.
Materials
Reagents
poly-D-lysine (SIGMA P6407 5 mg)
CYTOSKELETON BUFFER: 10 mM Pipes pH 6.8, 100 mM NaCl, 300 mM sucrose, 3mM MgCl2, 1mM EGTA, 0.5% Triton-X100
CYTOSKELETON STRIPPING BUFFER: 10 mM TrisHCl pH 7.4, 10 mM NaCl, 3mM MgCl2, 1% Tween 40(v/v), 0.5 % sodium deoxycholate (v/v)
FIXATIVE: 150mM 2-bromo-2nitro-1,3 propanediol (SIGMA B0257), 108 mM diazolidinyl urea (SIGMA D5146), 10 mM NaCitrate, 50 mM EDTA, pH5.7
PERMEABILIZATION BUFFER: 100 mM Tris HCl pH 7.4, 50 mM EDTA, 0.5% TRITON-X-100
PBG 10x STOCK SOLUTION: 5% BSA, 2% of gelatin from cold water fish skin (SIGMA cat. G-7765) in PBS 1x
DAPI ( SIGMA cat.D-9564)
Mowiol mounting medium ( CALBIOCHEM cat. 475904)
Primary Antibodies: RPA /p34 (Neomarkers Cat. #MS-691-P1); 1:300
ATR goat (Santa Cruz Biotechnologies N-19) 1:50
Equipment
Procedure
1)Prepare poly-D-lysinated coverslips (poly D lysine 50μg/ml)
2)Plate 15-20×103 cells on coverslips 1 day before the staining
Preextraction and fixation
3)Wash cells grown on coverslips gently 2x with ice-cold PBS 1x
4)Treat the cells with ice-cold cytoskeleton buffer 5’ on ice
5)Aspirate and add an ice-cold cytoskeleton stripping buffer for 5’ on ice
6)Wash 3x with ice-cold PBS
7)Fix the cells in fixative for 30’ RT
8)Wash in 2x PBS 1x
9)Permeabilize for 15’ at RT with permeabilization buffer
10)Wash 3x in PBS 1x
Blocking and Staining
11)Block cells for 30’ with PBG 1X
12)Stain with primary antibody diluted in PBG 1X at indicated concentration for 1h at RT
13)Wash cells gently 3X5’ in PBG 1X
14)Stain with secondary antibody diluited in PBG 1X for 1h at RT
15)Wash cells gently 2X5’ in PBG 1x and 2X5’ in PBS 1X
16)Stain with Dapi for 5' in the dark
17)Mount the coverslips with 3 μl of Mowiol mounting medium
18)Let the coverslips dry before microscope analysis
Troubleshooting
Higher possibility to lose cells because of different steps. Always use poli D-lysinated coverslips especially for RAS-induced senescent cells
Critical Steps
Anticipated Results
References
Mirzoeva, O. K. & Petrini, J. H. J. DNA Damage-Dependent Nuclear Dynamics of the Mre11 Complex. Mol. Cell. Biol. 21, 281-288 (2001).
Acknowledgements
Keywords
ΑΤR, ATRIP, RPA, replication stress,
Immunofluorescence,
DNA damage checkpoints,
DNA damage response,
Oncogene-induced senescence
Figure 1
Confocal analysis of oncogene-induced senescent cells immunostained for RPA/ATR/ATRIP complex by indirect immunofluorescence with preextraction
