FISH with #1.5, 40 mm coverslip

1. Sonicate coverslips: 10’ in 1:12 Branson solution, 10’ de-ionized water, 10’ Ethanol, and dry, or leave immersed in EtOH. Air-dry in the hood before placing in a tissue-culture dish.

Seeding:

1. Place 7 40 mm coverslips in a big petri dish and cover with ~20 ml media.
2. Trypsinize cells and resuspend in 5 ml (1 ml cells + trypsin and 4 ml fresh media).
3. Use 1 ml for passaging, and 4 ml for seeding. Add the 4 ml to the big petri dish with the 7 coverslips.
4. The plate should be confluent in ~ 4 days.

Fix:

1. Aspirate media, and add ~20 ml PBS.
2. Aspirate PBS, and add a 15-20 ml fix solution (4% formaldehyde in PBS).
3. Incubate for 10′ on a Roto-Mix.
4. Aspirate, wash with ~20 ml PBS.
5. Aspirate, add 20 ml PBS and store in cold room.

Permeabilization:

1. Aspirate, add 15-20 ml permeabilization solution [0.5% (v/v) Triton-X-100 in 1X PBS].
2. Incubate for 10′ on a Roto-Mix.
3. Aspirate, wash with ~20 ml PBS.

FISH:

• 2X SSCT has 0.1% Tween, which means 4x SSCT should have 0.2% Tween.

*Unless mentioned otherwise, all reactions are carried at RT. All RT incubations are carried on top of a platform shaker. Pre-heat one water bath to 60c, and pre-warm ~6 ml 2X SSCT+ 50% formamide to 60c. Pre-heat another water bath to 80c. When pipetting, pipette to the side of the tissue-culture dish, and not directly onto the coverslips.

1. Place two 40 mm coverslips, each  in a small glass-bottom tissue-culture dish (for instance: P50G-1.5-30-F; MatTek), and add ~3 ml PBT per plate (coverslip). Incubate for 2′.
2. Aspirate, add ~3 ml 0.1N HCl, and incubate for 5′.
3. Aspirate, add ~3 ml 2X SSCT, and incubate for 1′.
4. Aspirate, add ~3 ml 2X SSCT, and incubate for 1′.
5. Aspirate, add ~3 ml 2X SSCT plus 50% formamide solution (not pre-heated), and incubate for 2′.
6. Place at glass-bottom petri dish, or coplin jar lid (and cover with para film), and cover with pre-heated 2X SSCT plus 50% formamide solution, and incubate at 60c water bath for 20′ (on top of a heat block).
7. Air-dry coverslips by leaning on a coplin jar while preparing hybridization mix. Place coverslips on a glass plane (preferably thin). Add 35 µl hybridization mix (50% formamide, 25% of 40% dextrane plus 8X SSCT solution, ~1.4 µM probe per 1 Mb, 1.4 µl RNAse A, and ultra pure water) on top of the center of each coverslip, and sandwich with a 22*30 mm #1.5 rectangular slide. Seal with rubber cement, and let dry at 37c oven for ~ 9 minutes.
8. Place the glass plane with coverslips on top in 80c water bath on top of a heat block. Denaturate for 3 minutes.
9. Transfer coverslips to a small glass-bottom tissue-culture dish.
10. Prepare hybridization chamber: place wet paper towels in a plastic box (e.g. microwave box). Place a thin rack to separate the sample from the paper towels, and place coverslips (samples) inside glass-bottom petri dish . Close plastic box to seal hybridization chamber. Place in 47c for ~2-3 nights.
11. Pre-warm 35 ml of 2X SSCT solution in 60c water bath.
12. Remove the rectangular coverslip and place coverslips in a glass bottom dish. Add 2X SSCT.
13. Aspirate, and wash 4 times with 2X SSCT at 60c for 5′ each time.
14. Aspirate and wash twice with room temp 2X SSCT, for 2′ each time.

If you plan to continue to imaging then, please click this link to the rest of this protocol: Day-of-experiment protocol, including fluidics

If you plan to include immunostaining, click this link: IF protocol following FISH

Thank you,

Guy