FISH on ibidi channel slides

Updated: 12/21/2023

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.

FISH steps were adapted by Gabrielle Dewson from Brian Beliveau and Hiroshi Sasaki at Peng Yin’slaboratory.

Recipes:

4% PFA (Make Day Of): Stock 16%

3.125 mL 16% PFA, 1.25 mL 10x PBS, 8.125 mL MilliQ H₂O

4x SSCT: Stock 20x SSC need 40 mL

            80 mL Tween + 8 mL 20x SSC + 31.9 mL MilliQ H₂O

2x SSCT:

            20 mL 4x SSCT + 20 mL MilliQ H₂O

4x SSC: Stock 20x SSC need 40 mL

            8 mL 20x SSC + 32 mL MilliQ H₂O

.5% Triton (Make Day Of): Stock 10% need 1 mL

            50 mL Triton in 950 mL PBS

Hybridization solution (Make Day Of):

            50 mL Formamide (chem hood) + 25 mL pre-made Hybe soln + 4 mL RNase A + 2 mL of 200 mM probe(s)+ 19 mL* MiliQ H₂O (for 1 probe) or 17 mL* MiliQ H₂O (for 2 probes) *total volume is 100 mL

2x SSCT + 50% Formamide (Make Day Of): Need 2 mL

            1 mL of 4x SSCT + 1 mL of Formamide

Wash Buffer (Make Day Of): need 10 mL

            5 mL 4x SSCT + 3.5 mL formamide + 1.5 mL H₂O

Gold urchins (fiducials):

4 mL of urchins in 116 mL 1x PBS

Beads:

1 mL for sonication, dilute in 499 mL PBS and then take 20 mL of the 1:500 solution in 180 mL PBS

PCA/PCD imaging buffer (wide field use only): Need 1 mL

15 mL 100x Trolox + 15 mL 100x PCD + 40 mL 40x PCA + 10 mL DAPI + 930m L PBS

Imaging Buffer GLOX from Bogdan Bintu (super res/tracing): Need 10 mL

2 mL 50% Glucose + 1 mL 20x SSC + 500 mL Tris-HCL + 6.2 mL MiliQH₂O + 100 mL BME + 200 mL Glox soln

Catalase:

.02 g in 1 mL of 1x PBS

Glox Soln (Make Day Of):

.02 g Glucose Oxidase + 125 mL 50 mM TRIS-50 mM NaCl + 75 mL Catalase

Vortex slightly and spin down

Add 1 mL of mineral oil on top (for tracing)

40% Dextran Sulfate solution:

4 g Dextran sulfate in 10 ml 8X SSCT.

First, pour 10 ml of 8X SSCT (4 ml 20X SSCT and 6 ml UPW)

Add 4 g Dextran Sulfate

Raise temperature to 65ºC.  

1X PBST

PBS + 0.1% Tween-20

36% HCl (conc. HCl)

12 N. 100 mL 8N HCl  = 66.67 mL 12 N HCl + 33.33 mL miliQ water.

50% D-glucose (Dextrose) (200 mL)

Pour 100 mL ddH2O into a beaker, heat, and start stirring.

When water is warm, add 100 gram dextrose

Wait a few minutes for the dextrose to go into the solution.

Add 100 mL ddH2O. Store at 4C.

RBS 10%:

50 mL RBS 50 in 450 mL of miliQ water

Seeding (channels 2-5):

1. Add 10⁶cells/mL for the entire slide (about 200 mL per well), which should result in confluency in the next day.
   1. For a single channel slide- 10⁶ cells per channel about 200 mL for the well
2. Fill channels 1&6 with PBS or media. These two channels will be used for calibration with tetraspeck beads.

Fix cells (channels 2-5):

1. Aspirate medium from all reservoirs using a vacuum trap.
2. Wash cells with PBS by slowly applying 130 mL into one empty reservoir of each channel and aspirating from the opposite reservoir for each channel. Don’t aspirate the entire channel volume.
3. Fix cells with 130 mL of 4% paraformaldehyde in PBS for 10min
4. Wash cells with 130 mL PBS, don’t let channel dry
5. Wash cells again with 130 mL PBS as described above.
6. Aspirate PBS from reservoirs
7. Add 130 mL PBS, cover with parafilm and lid
8. Store in cold room for up to two weeks.

Permeabilize (channels 2-5):

1. Turn on the water baths!
2. Apply 130 mL of 0.5% Triton® X-100 in PBS for 10 min.
3. Wash cells twice with PBS.

DNA FISH:

* Day 2: 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.

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

*Channels 1&6, with no cells, can be kept in PBS until step 21.

1. Aspirate reservoirs, add 130 mL 1X PBST and incubate 2’ RT.
2. Aspirate, add 130 mL 0.1N HCl, and incubate 5’ RT.
3. Aspirate, add 130 mL 2X SSCT, and incubate 1’ RT.
4. Aspirate, add 130 mL 2X SSCT, and incubate 1’ RT.
5. Aspirate, add 130 mL 2X SSCT + 50% (vol/vol) formamide and incubate 2’ RT
6. Aspirate, add 130 mL 2X SSCT + 50% (vol/vol) formamide and incubate for 20’ at 60ºC.
   1. Seal slide with parafilm and lid. Place slide on a heat-block. 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 mL hybridization solution
   1. [50 mL formamide, 25 mL hybe mix containing 8X SSCT and 40% Dextran Sulfate, 4 mL of 10 μg/μl RNase A, 2 mL of 200 µM probe (per 20-25,000 oligos), 1 mL of 10 mg/mL yeast tRNA (100 ug/mL final) and add UPW to 100 mL].
8. Denature for 3’ at 80ºC in a water bath on top of a heat block.
9. 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 42ºC (can be left for up to 2 days).
10. Remove the parafilm and wash with 2X SSCT.
11. Wash for 5’ in pre-warmed 2X SSCT at 60ºC.
12. Aspirate, wash for 5’ in pre-warmed 2X SSCT at 60ºC. In the meantime, degas sonicator. Take 1 mL of 100 nm tetraspeck beads and sonicate for 10 minutes. After sonication, dilute 1:5,000 in PBS.
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 2’ in 2X SSCT at RT.
16. Aspirate, wash for 2’ in 2X SSCT at RT.
17. Wash with PBS.
18. Add 1:40 90 nm gold nano-urchins in PBS. Centrifuge at 500g for 3 minutes.
19. Channels 2-5: Hybe secondaries for 30 minutes in RT (2X SSC, 0.03% Tween-20, 10 mL of 50% (wt/vol) dextran sulfate (final concentration 5%), 30% formamide, 1 mL of 10 mg/mL yeast tRNA (final concentration 100 ug/mL) and 500 nM of secondaries).
20. 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).
21. Wash channels 2-5 with 2X SSCT + 35% formamide 5’ at RT. Repeat three times. Wash channels 1&6 with PBS. Total washes: 3-5 depending on background noise level.
22. Aspirate, wash for 2’ in PBS at RT.
23. Exchange PBS and store in PBS at 4ºC.
24. Aspirate reservoirs, and then add 130 mL imaging buffer.