FISH on ibidi channel slides

Updated: 03/31/2022

For super-resolution, you can choose to either glue your choice of coverslips (preferably 1.5H) to a sticky channel slide or use the premade channel slide VI 0.5. For fluidics applications use the u-slide I Luer.

Constructing the channel (if using sticky-slides):

1. Sonicate coverslips 10 minutes in 1:11 Branson solution, then rinse thoroughly, sonicate 5 minutes in deionized water, and 10 minutes in IPA or Ethanol. You can store them in a Coplin jar filled with EtOH, or dry and continue to the next step.
2. Assemble channels in the tissue culture hood, by adhering a cover slip to the bottomless sticky-slide. Press it with Q-tips, and incubate at 20-40c overnight. It is recommended to press it when incubating overnight. Can also use in conjunction with ibidi clamp.

Seeding (channels 2-5):

Add 2-5*10⁵ cells/ml in channels 2-5 for standard culturing. Alternatively, add 10⁶ cells/mL, which should result in confluency in the next day. Fill channels 1&6 with PBS or media. These two channels will be used for calibration with tetraspeck beads.

FISH steps were adapted from Brian Beliveau and Hiroshi Sasaki at Peng Yin’s laboratory.

Fix cells (channels 2-5):

1. Aspirate medium from all reservoirs using a cell culture aspiration device. Wash cells with DPBS by slowly applying 130 µl into one empty reservoir of each channel and aspirating from the opposite reservoir for each channel. Don’t aspirate the entire channel volume.
2. Fix cells with 130 µl of 4 % paraformaldehyde in PBS. After 10 min flush the liquid inside the channel by filling one well with 130 µl PBS and removing the content of the reservoir from the other well; ensuring the channel is never dry.
3. Wash cells again with 130 µl PBS as described above.
4. Aspirate PBS rom reservoirs, add 130 µl PBS, cover with parafilm and lid, and store in cold room foor up to two weeks.

Permeabilize (channels 2-5):

1. Apply 130 µl of 0.5% Triton® X-100 in PBS for 10 min.
2. Wash cells twice with PBS.

DNA FISH:

* pre-warm 1 ml 2X SSCT + 50% (vol/vol) formamide at 60c. Also prepare 1 ml 2X SSCT + 50% (vol/vol) formamide and leave at RT.

2X SSCT = 2X SSC + 0.1% Tween-20

*Warm washes and denaturation are done on top of a heat block inside water baths.

*Channels 1&6 can be kept in PBS until step 21.

1. Aspirate reservoirs, add 130 µl 1X PBT and incubate 2’ RT.
2. Aspirate, add 130 µl 0.1N HCl, and incubate 5’ RT.
3. Aspirate, add 130 µl 2X SSCT, and incubate 1’ RT.
4. Aspirate, add 130 µl 2X SSCT, and incubate 1’ RT.
5. Aspirate, add 130 µl 2X SSCT + 50% (vol/vol) formamide and incubate 2’ RT
6. Aspirate, add 130 µl 2X SSCT + 50% (vol/vol) formamide and incubate for 20’ at 60ºC. Seal slide with parafilm and lid. Place slide on a heat-block, covered with a thin layer of deionized water. Make sure there is no overflow of water, which can lead to water penetrating the reservoirs.
7. Aspirate reservoirs and channel, and then quickly add 100 µL hybridization solution [50 µL formamide, 25 µL hybe mix containing 8X SSCT and 40% Dextran Sulfate, 4 μl of 10 μg/μl RNase A, 2 µL of 200 µM probe (per 20-25,000 oligos) and add UPW to 100 µL].
8. Cut and place parafilm on the inside of the lid. Cut another piece of parafilm and tape around the upper end of the reservoirs and stretch on top of the lid. Add weight (e.g., rubber stop) to press the lid while denaturing.
9. Denature for 3’ at 80c in a water bath on top of a heat block.
10. Wrap and seal the entire ibidi slide with a parafilm. Place the wrapped ibidi slide on top of a 96-well PCR rack. Place wet paper towels inside a plastic box and place the rack with an ibidi slide inside. Close the plastic box and hybridize overnight at 42c.
11. Remove the parafilm and wash with 2X SSCT.
12. Wash for 5’ in pre-warmed 2X SSCT at 60c.
13. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60c. In the meantime, degas sonicator. Take 1 µL of 100 nm tetraspeck beads and sonicate for 10 minutes. After sonication, dilute 1:5,000 in PBS.
14. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60c.
15. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60c.
16. Aspirate, wash for 2’ in 2X SSCT at RT.
17. Aspirate, wash for 2’ in 2X SSCT at RT.
18. Wash with PBS.
19. Add 1:40 90 nm gold nano-urchins in PBS. Centrifuge at 500g for 3 minutes.
20. Channels 2-5: Hybe secondaries for 60 minutes in RT (2X SSC, 0.03% Tween-20, 5% dextran sulfate, 30% formamide, and 500 nM of secondaries).
21. Channels 1&6: Aspirate entirely. Add the 1:5,000 100 nm tetraspeck beads in PBS solution you previously made. Incubate for ~60 minutes (same time you incubate channels 2-5).
22. Wash channels 2-5 with 2X SSCT + 35% formamide 5’ at RT. Repeat three times. Wash channels 1&6 with PBS.
23. Aspirate, wash for 2’ in PBS at RT.
24. Exchange PBS and store in PBS at 4c.
25. Aspirate reservoirs, and then add 130 µl imaging buffer.