Berglund:Cell Culture FISH protocol: Difference between revisions
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Latest revision as of 10:14, 17 December 2014
FISH Protocol
Prep coverslips
Materials needed:
Coverslips
1M HCl
Petri dish with lid
Fine tweezers
Polylysine
1X PBS
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:
1X PBS
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
2X SSC
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
Get someone to train you how to use the confocal and collect images