Materials and Reagents

Cells
4% PFA
Methanol/acetone 1:1
TritonX100
Primary antibody : 53BP1 #NB 100-304 rabbit from Novus
Second antibody: goat anti-rabbit Alexa Fluor^® 488 Dye
PBS
Glycine
Fish gelatin (Sigma-Aldrich, catalog number : G7041 )
BSA
Formamide
Tris HCl, pH 7.4
Telomeric PNA probe (TelC-Cy3 from PANAGENE, catalog number: F1002-5 )
DAPI
Mowiol 4-88 reagent (Calbiochem^®)
PBG (see Recipes)
Hybridization mixture (see Recipes)
Blocking reagent (Roche Diagnostics, catalog number: 11096176001 ) (see Recipes)
Wash solution I (see Recipes)
Wash solution II (see Recipes)

Equipment

Glass coverslips
12 multiwell plate
Metal thermoblock
Humidified chamber

Procedure

Grow cells on glass coverslips (e.g. BJ normal human fibroblasts).
Transfer the coverslip to a 12 multiwell plate.
Wash briefly with 1x PBS.
Fix with either 4% PFA, 10 min, RT or methanol/acetone 1:1, 2 min, RT (it depends on the antibody, does not affect the FISH signal; use methanol/acetone for 53BP1 staining).
Wash with 1x PBS, 3 times, 5 min.
Only for PFA-fixed cells, incubate with 0.2% TritonX100 in PBS, 10 min, then wash with 1x PBS, 3 times, 5 min.
Block with 1x PBG, 1 h, RT.
Incubate with primary antibody diluted in 1x PBG, 50 μl for each coverslip. Incubation time depends on the antibody, most work in 1 h, RT, or overnight at 4 °C. (For 53BP1 dilute 1:200 and incubate 1 h at RT).
Wash with 1x PBG, 3 times, 5 min.
Incubate with secondary antibody diluted in 1x PBG, 45 min, RT.
Wash with 1x PBG, twice, 5 min.
Wash with 1x PBS, twice, 5 min.
Re-fix cells with PFA 4% + triton 0.1%, 10 min RT (use PFA also if you have previously fixed cells with methanol/acetone).
Incubate with glycine 10 mM in H₂O, 30 min, RT.
Wash with 1x PBS, 3 times, 5 min.
Prepare the hybridization mixture and put 20 μl on a glass slide for each coverslip.
Transfer the coverslip carefully on the drop without making bubbles.
Put the slide directly on a metal thermoblock at 80 °C, 5 min.
Hybridize in a humidified chamber, 2 h, RT.
Remove coverslip from the slide and put it back in the 12 wells plate.
Wash with Wash solution I, twice, 15 min.
Wash with Wash solution II, 3 times, 5 min.
Incubate with DAPI, 2 min, RT.
Wash briefly with 1x PBS.
Mount with mowiol.
Store the slides at 4 °C for short time storage (2 weeks) or at -20 °C. It is recommended to analyze the fluorescence as soon as possible to avoid fluorophore fading.

Figure 1. An image of ImmunoFish stained human fibroblasts cells. DAPI is in blue, 53BP1is in green and telomeric PNA probe is in red.

Recipes

10x PBG (prepare 5 ml aliquotes and store them in 50 ml tubes at -20 °C, the day of immunoFISH dilute them in 1x PBS)

Fish gelatin
2%

BSA
5%

1x PBS
to volume
Hybridization mixture (always prepare fresh)

Formamide
70%

Blocking reagent
1x

Tris HCl pH 7.4
10 mM

Telomeric PNA probe
0.5 μM

H₂O
to volume
10x Blocking reagent
Prepare small aliquots and store them at -20 °C
Wash Solution I (250 ml) (always prepare fresh)

Formamide
175 ml

BSA 10%
2.5 ml

Tris HCl 1 M pH 7.4
2.5 ml

H₂O
to volume
Wash Solution II (350 ml) (always prepare fresh)

Tris HCl 1 M pH 7.4
35 ml

NaCl 5 M
10.5 ml

Tween 20 10%
2.5 ml

H₂O
to volume

Acknowledgments

The F.d’A.d.F. laboratory is supported by FIRC (Fondazione Italiana per la Ricerca sul Cancro), AIRC (Associazione Italiana per la Ricerca sul Cancro), European Union (GENINCA, contract number 202230), HFSP (Human Frontier Science Program), AICR (Association for International Cancer Research), EMBO Young Investigator Program and Telethon.

References

Fumagalli, M., Rossiello, F., Clerici, M., Barozzi, S., Cittaro, D., Kaplunov, J. M., Bucci, G., Dobreva, M., Matti, V. and Beausejour, C. M. (2012). Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation. Nat Cell Biol 14(4): 355-365.