McClean:FISH

This is not a final protocol and almost certainly requires further refinement.

Protocol

Fixing Cells

1. Grow culture to OD600= 0.4-0.8 in YPD or SD Medium (Approx. 120 Klett)
2. Fix cells by adding 32% paraformaldehyde directly to the culture to a final concentration of 3.2% (900 μL culture + 100μL paraformaldehyde). Keep at room temperature for 45 minutes
   • If the cells are not dense you can take a sample of 1.8 μL cells in a 2ml eppendorf, or an even greater volume. If a larger sample, after the 45 minutes spin the cells down at 3000Xg for 5 minutes, resuspend in buffer B and transfer to a 1.5 or 2 ml eppendorf.
3. Wash cells 3X with 1ml Buffer B, spinning at 3000Xg for 5 minutes each time.
4. Resuspend pellet in 1ml spheroplast buffer plus VRC plus lyticase plus BME (890ul Buffer B + 100ul VRC + 10ul Lyt + 2ulBME).
5. Incubate at 37C - Monitor digestion: ~90% phase dark (digested) cells.
6. Spin 5 min at 2000rpm (do not spin too hard as cells are fragile due to lyticase).
7. Wash 3X with Buffer B, spinning at 2000 rpm for 5 minutes.
8. Gently add 1ml 70% ethanol.
9. Incubate at -20C for several hours or overnight. (If you are in a hurry you can incubate at 4C for 1 hour instead.)

• At this point the cells can be stored at -20C for several weeks or months

Hybridization for samples adhered to coverglass NOTE: Can try to adapt the protocol to work for 96-well plates

1. Mix a small volume of the probe stock (1-10 µL) with Hybridization Solution to 100 µL final volume for each sample to be hybridized. Vortex and centrifuge the probe solution(s) briefly. For the initial test of a probe-set it is best to start 4 separate hybridization reactions to determine which dilution of the probe stock is optimal for detection. Add 1 µl from each of each of the working dilutions (1:10, 1:5, 1:2.5 ) as well as the probe stock itself into separate aliquots of the hybridization solution.
2. Gently Aspirate the 70% ethanol off of the sample.
3. Add 1 mL of wash buffer with the same percentage formamide (10%) as the hybridization buffer and let stand for 2-5 minutes.
4. Aspirate the wash buffer and then add 100 µL of hybridization solution plus probe.
5. Place a carefully cleaned coverslip over the sample to prevent drying of the hybridization solution during the incubation.
6. Incubate in a dark humidified chamber for 4 hours at +37 °C. If stronger signals are desired for low expressed genes, the hybridization can be done overnight.
7. Add 1 mL of wash buffer to the sample, remove the coverslip very carefully so as not to disturb the cells underneath.
8. Incubate at +37 °C for 30 minutes in the dark.
9. Aspirate the wash buffer, then re-suspend in another 1 mL of wash buffer with 5 ng/mL DAPI for nuclear counterstaining.
10. Incubate at +37 °C for 30 minutes in the dark.
11. If you are imaging without using glucose oxidase (GLOX) anti-fade, then add 1 mL of 2x SSC and proceed to imaging. If you are imaging with the GLOX anti-fade solution, aspirate the buffer and resuspend in 2x SSC.
12. Aspirate the SSC and add the GLOX buffer without enzymes for equilibration; incubate for 1-2 minutes.
13. Aspirate the buffer and re-suspend in the 100 µL of GLOX buffer to which the enzymes (glucose oxidase and catalase) have been added.
14. Place a carefully cleaned coverslip over the sample to spread the GLOX buffer over the entire sample and slow evaporation.
15. Proceed to imaging

Solutions

Buffer B

• 1.2M Sorbitol
• 100mM KHPO4 pH 7.5

e.g. 1MKH2PO4 8ml

1MK2HPO4 41.5ml

Sorbitol 109.3g

Lyticase

Resuspend Sigma cat #L5263 in 1X PBS to 25000U per ml. Store at -20C in single use aliquots (50U). Use 50U in 1ml of spheroplast buffer for slow growing cells.

PBS 1X (purchased)

Hybridization Buffer (SingleMoleculeFish.com)

• Dextran sulfate (1 g), catalog #D6001 from Sigma or equivalent
• 20X saline-sodium citrate (SSC) (nuclease-free) (1 mL), catalog #82021-484 from VWR or equivalent
• Formamide (deionized) (1 mL for 10% final concentration), catalog #AM9342 from Ambion or equivalent
• Nuclease-free water (to 10 mL final volume), catalog #AM9932 from Ambion or equivalent

WARNING: Formamide is a teratogen that is easily absorbed through the skin and should be used in a chemical fume hood. Be sure to let the formamide warm to room temperature before opening the bottle.

Wash buffer without DAPI (50 mL):

• 20X SSC (5 mL), catalog #82021-484 from VWR or equivalent
• Formamide (deionized) (5 mL for 10% final concentration), catalog #AM9342 from Ambion or equivalent
• Nuclease-free water (to 50 mL final volume), catalog #AM9932 from Ambion or equivalent

Wash buffer w/DAPI (50 mL):

• DAPI, catalog #D9564 from Sigma or equivalent (concentration is at SingleMolecularFish.com)
• 20X SSC (5 mL), catalog #82021-484 from VWR or equivalent
• Formamide (deionized) (5 mL for 10% final concentration), catalog #AM9342 from Ambion or equivalent
• Nuclease-free water (to 50 mL final volume), catalog #AM9932 from Ambion or equivalent

Anti-fade buffer and enzymes (“GLOX” buffer):

• 10% glucose in nuclease-free water, catalog #158968 from Sigma or equivalent
• 1 M Tris-HCl, pH 8.0, catalog #AM9855G from Ambion or equivalent
• 20X SSC, catalog #82021-484 from VWR or equivalent
• Nuclease-free water, catalog #AM9932 from Ambion or equivalent
• Glucose oxidase (diluted to 3.7 mg/mL stock), catalog #G0543 from Sigma or equivalent
• Catalase, catalog C3155 from Sigma or equivalent

NOTE: Mix together 0.85 mL of nuclease-free water and add 100 µL of 20X SSC, 40 µL of 10% glucose and 10 µL of 1 M Tris-HCl. Vortex and then transfer 100 µL of this “GLOX” buffer to another tube, to which one should add 1 µL of the glucose oxidase stock and 1 µL of mildly vortexed catalase suspension. The remainder can be used as an equilibration buffer.