Immuno-FISH

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.

Reagents

General:

• Purified FISH probes. 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
• 0.01% sodium azide in PBS
• 1% sodium azide in water
• Antibody buffer: 3% BSA in PBS
• Antibody buffer with 1% Triton X-100
• Antibody wash buffer: antibody buffer diluted 1:10 with PBS + 0.1% Triton X-100

Denaturation Solutions

• 20X SSC stock: 3M NaCl, 300 mM sodium citrate
• Denatuation solution: 70% deionized foramide in 2X SSC
• Detection solution: 1% BSA + 0.05% TWEEN 20 in 4X SSC
• Detection wash: 0.05% TWEEN 20 in 4X SSC
• Hybridization solution: 50% deionized foramide, 10% dextran sulfate in 2X SSC
• Hybridization wash: 50% deionized foramide in 2X 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 buffer + 1% Triton X-100.
6. Wash 1 x 10 min with antibody buffer.
7. Add primary antibody to slide 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 wash buffer.
9. Repeat steps 4-8 with secondary antibody (if needed).
10. It may be a good idea to confirm antibody labelling before moving onto FISH staining.