McClean: FISH (Ping)

Overview

This is a draft protocol. This will be optimized to measure transcriptional response from the pheromone and hyperosmotic stress pathway in S. cerevisiae

Solutions

Buffer B

• 1.2M Sorbitol
• 100mM KHPO₄ pH 7.5

To make mix together:

• 8ml of 1M KH₂PO₄
• 41.5 ml of 1M K2₂HPO₄
• Sorbitol 10.93g

Lyticase

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

PBS 10X (RNase free)

Ambion #AM9624 or equivalent

Nuclease Free Water

Ambion #AM9932 or equivalent

Hybridization Buffer

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

Mix together 1g of Dextran sulfate, 1ml of 20x saline-sodium citrate (SSC), 1ml of formamide and bring everything up to 10ml with nuclease-free water.

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, catalog #82021-484 from VWR or equivalent
• Formamide (deionized), catalog #AM9342 from Ambion or equivalent
• Nuclease-free water, catalog #AM9932 from Ambion or equivalent

Mix together 5ml of SCC, 5 ml of formamide (for 10% final concentration), and bring everything up to 50ml with nuclease-free water.

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 w/DAPI (50 mL)

• DAPI, catalog #D9564 from Sigma or equivalent (Stock is at?)
• 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

Add together 5ml SSC, 5ml of formamide (for a final concentration of 10%), add DAPI to have a final concentration of 5ng/ml and then bring volume up to 50ml with nuclease-free water.

10% glucose

• α-D-Glucose, catalog #158968 from Sigma or equivalent
• Nuclease-free water, catalog #AM9932 from Ambion or equivalent

Weight out 10g of glucose into 70 ml of nuclease-free water. Dissolve glucose and bring volume up to 100ml with nuclease-free water. Autoclave.

Anti-fade buffer WITHOUT enzymes

• 10% glucose in nuclease-free water
• 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.

Anti-fade buffer WITH enzymes (“GLOX” buffer)

• 10% glucose in nuclease-free water
• 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.

Protocol

Fixing Cells

1. Grow culture to approximately Klett 120 in LFM media (can also use YPD or SC media). If you are measuring OD, grow cells to an OD of ~0.4-0.6
2. Fix cells by adding 32% paraformaldehyde directly to the culture to a final concentration of 3.2% (12ml culture + 3ml paraformaldehyde). Incubate at room temperature for 45 minutes.
3. Spin the cells down at 3000Xg for 5 minutes. Resuspend in 1.8ml Buffer B and transfer to a 2 ml eppendorf.
4. Wash cells 3X with 1.8ml Buffer B, spinning at 3000Xg for 5 minutes each time.
5. Resuspend pellet in 1002 μL spheroplast buffer plus VRC plus lyticase plus BME (890ul Buffer B + 100ul VRC + 10ul Lyt + 2ulBME).
6. Incubate at 37°C. Monitor the digestion every 10 minutes or so. Check the cells on the small scope by the nanodrop. Once you reach ~90% phase dark (digested) cells the digestion is done. The first time you try the protocol, this step will need to be optimized. Previous attempts suggest that about 15 minutes works well.
7. Spin 5 min at 3000xg (do not spin too hard as cells are fragile due to lyticase).
8. Wash 3X with 1ml Buffer B, spinning at 2000 rpm for 5 minutes each wash.
9. Gently add 1ml 70% ethanol.
10. Incubate at 4°C overnight. Cells can be stored like this for about a week before hybridization (perhaps longer, this would need to be tested. The Botstein lab protocol seems to indicate that cells can be stored in this state at -20°C for up to several months).
11. At this point, it would be a good idea to coat the bottom of a 96-well glass-bottomed plate with 30μL of concanavalin A (see Concanavalin A for Microfluidics) and incubate overnight to increase cell adherence for imaging. Cover the top of the plate wells with lab tape or similar to prevent evaporation.

Hybridization

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. Incubate the yeast cells in 1ml of 0.1mg/mL concanavalin A (see Concanavalin A Solution (without ions) ) for 2 hours and then let settle onto the chambered coverglass pre-coated with concanavalin A. The volume of the wells of the 96-well plate is a maximum of 100μL, so add 100μL of cells, and let this settle. Depending on the density of cells this may be too much or too little and the amount of settling time or the volume of cells added may need to be adjusted.
4. Add 100μL of wash buffer with the same percentage formamide (10%) as the hybridization buffer and let stand for 2-5 minutes.
5. Aspirate the wash buffer and then add 100 µL of hybridization solution plus probe.
6. Cover the wells with parafilm or tape during incubation to prevent drying of the hybridization solution during the incubation.
7. 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.
8. Add 100μL of wash buffer to the sample.
9. Incubate at +37 °C for 30 minutes in the dark.
10. Aspirate the wash buffer, then re-suspend in another 100 mL of wash buffer with 5 ng/mL DAPI for nuclear counterstaining.
11. Incubate at +37 °C for 30 minutes in the dark.
12. Aspirate the wash buffer and resuspend in 2x SSC.
13. Aspirate the SSC and add the GLOX buffer without enzymes for equilibration; incubate for 1-2 minutes.
14. Aspirate the buffer and re-suspend in the 100 µL of GLOX buffer to which the enzymes (glucose oxidase and catalase) have been added.
15. Proceed to imaging