Immuno-FISH

Note: this is a protocol in development. This is a protocol for performing Fluorescence In-Situ Hybridization alongside immunostained cells. The protocol assumes that the cells are already grown on a coverslip, treated, and are ready for imaging. Note that this protocol uses an ethanol dehydration/denaturation step, and as such is unlikely to work with fluorescent proteins. This protocol also assumes that you have directly labelled (e.g. fluorescent) FISH probes. Protocol is modified from Ye at al (2009).

Reagents

General:

• Purified FISH probes, ~25 uM. Typically, a small plasmid (pluescript) bearing 2-3 kb of the target region of the genome, nick-labelled with the desired fluorphore and digested into 200-500 bp fragments.
  • If using a Jenna Kit:
    1. Process 1.5 μg of DNA & collect using a monarch kit into 50 μl
    2. Use a 1:2500 dilution of the labelled probes for staining
• Antibody(s) targeting proteins of interest.
• Secondary Fab's if the antibody is not directly labelled.
• 2% and 4% PFA in PBS
• Antibody buffer: 3% BSA in PBS
• Antibody high wash: antibody buffer with 1% Triton X-100
• Antibody low wash: high-wash buffer diluted 1:10 with PBS

Denaturation Solutions

• 20X SSC stock: 3M NaCl, 300 mM sodium citrate, pH 7.0
• Denatuation solution: 70% deionized foramide in 2X SSC
• Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC
• Hybridization wash: 50% deionized foramide in 2X SSC
• Detection wash: 0.05% TWEEN 20 in 4X SSC

Immunostaining

1. Immunostain as per our standard intracellular procedure.
2. After labelling and washing perform a light secondary fixation, 2% PFA in PBS, 10 min, room temperature.
3. Rinse 2x with PBS.
4. It may be a good idea to confirm antibody labelling before moving onto FISH staining.

FISH Staining

1. Dilute the FISH probe in hybridization solution, and heat at 75C for 10 min.
2. Incubate denatured probe at 37C for 10 min. During this time denature your coverslip (steps 3-4, below).
3. Denature the coverslip in denaturation solution, 70C, 2 min
4. Dehydrate the slide by immersing the coverslip in 75%, 90% then 100% ethanol, 2 min per immersion. Air dry slide.
5. Load the denatured probe onto the coverslip, cover/seal the slide (parafilm or other) and incubate overnight at 37C in a humidified chamber.
6. Uncover the coverslip and wash 3 x 5 min in 42C hybridization wash solution.
7. Wash 3 x 5 min in 2x SSC, 42C.
8. Wash 3 x 5 min in detection wash at room temperature.
9. Wash 1 x 10 min with PBS.
10. Counter-stain with DAPI or Hoescht, if desired.

Imaging

Prepared coverslips should either be imaged immediately in imaging buffer (rutin can be added to reduce photobleaching), or can be mounted on a slide using an anti-fade mounting solution and imaged at a later time.