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Protocols

FISH with ibidi sticky slides

Jan 14, 2021, 14:42 PM by Guy NIr

FISH with ibidi sticky-slide VI 0.4

  • Adapted from Brian and Hiroshi

sticky-Slide-VI-0.4

80608 Sticky-Slide VI 0.4: sterilized

FISH steps were adapted from Brian Beliveau and Hiroshi Sasaki from Peng Yin’s laboratory. http://molecular-systems.net/

Constructing the channel:
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:
1. Place µ-Slide in 37c incubator the night before.
2. Trypsinize cells and Resuspend in 5 ml (1 ml cells + trypsin and 3 ml fresh media).
3. Count cells (1 ml of 1:10 cells in PBS).
4. Resuspend an aliquot to a working solution of 5*105 cells/ml.
5. Introduce 130 µl of working solution to the channel, and incubate overnight at 37c, 5% CO2 incubator, cover with the lid.

Brian’s labteks: I take a 1:5 of the trypsinized cells, dilute that ~1:20 (500 µl of cells + trypsin into ~10 ml of fresh media), then seed 250 µl per well of the 1:20

Cover slips:
1. Sonicate coverslips: 5’ in 1:11 Branson solution, 5’ de-ionized water, 5’ IPA, and dry.
2. Following trypsinization, and 5’ centrifugation at 1200 rpm, Resuspend confluent plate in 4 ml.
3. Mark coverslips seeding plane (side), by writing ‘1’ in each corner.
4. Place cover slips on tips in a 10 µl tip box, with seeding plane facing upwards.
5. For a whole cover slip, use ~800 µl of cell suspension, and box. For ~3 channels, box with ~200 µl.
6. Leave overnight at 37c.

Fix and permeabilize:
1. Aspirate medium from all reservoirs using a cell culture aspiration device. Wash cells with Dulbecco´s PBS 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. Apply 130 µl of 0.5% Triton® X-100 in PBS for 10 min (25 µl Triton® X-100 in 5 ml PBS).
5. Wash cells with PBS.
6. Aspirate PBS rom reservoirs, add 130 µl PBS, cover with parafilm and lid, and store in cold room.

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.
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 50 µl hybridization solution (25 µl formamide, 12.5 µl hybe mix containing 8X SSCT and 40% Dextran Sulfate, 125 µM probe and UPW). Seal each reservoir with parafilm, place lid on the slide, and use tape seal the edges of the parafilm. Add weight to press sticky slides to coverslip while denaturating! You can also choose to increase hybridization volume to ~80 µl, which will reduce bubbles and evaporation.
8. Denature for 3’ at 80ºC.
9. Hybridize overnight at 47ºC. When you have many probes (I would say in the order of tens of thousands of probes), it is advisable to hybridize for longer duration. I have hybridized for over 72 hours without evaporation.
10. Remove parafilm from each reservoir.
11. Add 130 µl pre-warmed (60ºC) 2X SSCT, aspirate.
12. Wash for 5’ in pre-warmed 2X SSCT at 60ºC.
13. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60ºC.
14. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60ºC.
15. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60ºC.
16. Aspirate, wash for 2’ in 2X SSCT at RT.
17. Aspirate, wash for 2’ in 2X SSCT at RT.
18. Hybe secondaries 25 minutes in RT.
19. Wash with 2X SSC + 30% formamide 5’ at RT.
20. Aspirate, wash for 3’ in 2X SSCT + 40% Formamide at RT.
21. Aspirate, wash for 3’ in 2X SSCT + 40% Formamide at RT.
22. Aspirate, wash for 2’ in 2X SSC at RT.
23. Aspirate, wash for 2’ in 2X SSC at RT.
24. Aspirate entire channel and add orange or 488 Fiducial 1:1,000 in PBS+ for 30 seconds in RT, aspirate, and wash with 2X SSCT.
25. Store in 2X SSC in 4c.
27. Aspirate reservoirs, and then quickly add 130 µl imaging buffer.

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