Berglund:Cell Culture FISH protocol

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FISH Protocol

Prep coverslips

Materials needed:
1M HCl
Petri dish with lid
Fine tweezers
Tissue culture plates

Place coverslips in 1M HCl, cover container
Heat @ 55°C overnight
Wash coverslips in water until the pH is ~5
Place coverslips in petri dish with lid and cover with DI water
Sterilize coverslips and fine tweezers in UV light for 30 minutes
In the tissue culture hood, place a coverslip in each well of your plate
Add polylysine (1 mL for a six-well plate), incubate at 37°C 1 hr
Aspirate off polylysine, and add 1X PBS, incubate at 37°C 15 min

Plate and transfect cells

Follow protocol on Berglund lab wiki

Fix cells
Materials needed:
4% paraformaldehyde in 1X PBS

Wash cells with 1X PBS (1 mL/well in a 6 well plate)
Add 1 mL 4% paraformaldehyde in 1X PBS to each well
Incubate @ RT 15 min
Wash 2X with 1X PBS
Store in 1 mL 1X PBS @ 4°C

Permeabilize cells

Materials needed
0.5% triton X-100 in 1X PBS

Aspirate off liquid
Add 2 mL of triton and shake @ RT 5 min

Probe cells

Probe solution
1 ng/μL probe
30% formamide
2 μg/mL BSA
66 μg/mL yeast tRNA
2 mM vanadyl ribonucleotide complex

you’ll also need 1X SSC and 30% formamide in 2X SSC

pre-wash cells with 1 mL 30% formamide in 2X SSC

Shake @ RT for 5 min
Add 1 mL of probe solution to cells
Probe @ 37°C with shaking overnight (cover plate in foil)

Aspirate off probe and add 1 mL 30% formamide in 2X SSC
Shake @ 42°C for 30 min
Aspirate off liquid and add 1 mL 1X SSC
Shake @ RT for 30 min

Mount slides

Materials needed
Vectasheild mounting medium with DAPI (from Vector laboratories, Inc)
Microscope slides
Fine tweezers

Apply a small drop of vectasheild to a slide
Pick up coverslips with tweezers and place cell-side down on vectasheild
Let solidify overnight

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