PNA IF-FISH Protocol

Required Solutions
Blocking solution
(in PBS, store at -20ºC in 10 ml aliquots)

• 1 mg/ml BSA
• 3% goat serum
• 0.1% Triton X100
• 1 mM EDTA pH 8.0

Blocking reagent (Roche 11096176001)

• Stock is 10%, dissolved in maleic acid buffer:
• 100 mM maleic acid
• 150 mM NaCl
• Adjust pH to 7.5 (20°C) with NaOH. Store at 4ºC

Hybridizing solution

• Make fresh. Final concentrations in dH2O.
• 70% formamide (from deionized stock)
• 0.5% blocking reagent (from 10% stock)
• 10 mM Tris-HCl pH 7.2
• FITC-TelC (1:500-1000) or Tam-TelG (1:3000)

PNA probes (Custom from BioSynthesis, Lewisville Texas)

• Tamra-TelG: Tam-OO-TTAGGGTTAGGGTTAGGG 3’
• Stock concentration: 53 µM in dH2O
• Store at 4°C in the dark.
• For FISH analysis, use 1:3000-5000 dilution dH2O

• FITC-TelC: FITC-OO-CCCTAACCCTAACCCTAA 3’
• Stock concentration: 111 µM in dH2O
• Store at 4°C in the dark.
• For FISH analysis, use 1:500-1000 dilution dH2O

Washing solution

• 70% formamide
• 10 mM Tris-HCl pH 7.2

3000x DAPI

• Dissolve 0.5 mg/ml 4',6-diamino-2-phenylindole (Sigma D-9542) in H2O.
• Stable for at least one year at 4°C.

Embedding medium

• Dissolve 20 mg p-phenylene diamine (Sigma P-6001) in 2 ml 10xPBS by vortexing.
• Immediately add 18 ml glycerol, mix carefully without creating air bubbles. Do not vortex.
• Store in 1 ml aliquots at -70°C.

* or use ProLong Gold Antifade Reagent (Invitrogen #P36934)

IF-FISH Protocol

• Grow cells on cover slips to sub-confluence. Aspirate medium and wash in PBS.
• Fix the cells for 10 min at rt in 2% paraformaldehyde or for 10 min at -20ºC in 100% cold methanol.
• Wash 2 x 5 min in PBS.
• Wash 5 min in PBS (optional). Cells can be stored at 4ºC at this point, add 1000x stock NaN3 from 10% stock in H2O. Use caution, NaN3 is poisonous.
• Incubate cover slips for 30 min in blocking solution.
• Incubate 1 hour in primary antibody diluted in 50-100 µl blocking solution.
• Wash 3 x 5 minutes in PBS.
• Incubate 30 min in secondary antibody* diluted in 50-100 µl blocking solution.
• Wash 3x 5 minutes in PBS.
• Fix the cells on the cover slips for 5 min at rt in 2% paraformaldehyde.
• Wash 2x 5 minutes each in PBS.
• Dehydrate the cells in ethanol, consecutively 70%, 95%, 100% EtOH, 5 min each. Aspirate the ethanol completely and let the cover slips dry for a couple of minutes.
• Place a drop of hybridizing solution (60-80 ml) on each cover slip.
• Denature with hybridizing solution for 3-10 min at 70-80ºC by placing slides on heat block.
• Incubate in the dark for 2 hours at rt or over night at 4ºC.
• Wash 2 x 15 min in washing solution.
• Wash 3 x 5 min in PBS; add DAPI to the second wash.
• Air dry at rt for 10 minutes.
• Embed and seal.

* Notes:

• If using TelC telomeric probe, match with RRX-conjugated secondary antibody.
• If using TelG telomeric probe, match with Alexa 488-conjugated secondary antibody.
• If you wish to co-stain two proteins and telomeres, combine TelC with red Cy3 and Far-red Cy5 (weaker staining).