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.
• 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. Grow and stimulate cells as required on #1.5 thickness coverslips.
2. Fix with 4% PFA in PBS, 15 min, 37C.
3. Rinse 2X with PBS.
4. Wash 1 x 10 min with antibody buffer.
5. Wash 1 x 10 min with antibody high-wash buffer.
6. Wash 1 x 10 min with antibody buffer.
7. Add primary antibody to coverslip at the desired dilution in antibody buffer; incubate 1 hr at room temperature, or overnight at 4C.
8. Wash 3 x 10 min with antibody low-wash buffer.
9. Repeat steps 4-8 with secondary antibody (if needed).
10. Lightly fix in 2% PFA in pBS, 10 min, room temperature.
11. Rinse 2x with PBS.
12. 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.
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.