McClean: FISH (Gasch)

Overview

This protocol is very similar to the protocol used by Ping Xu and M. McClean for both the Jove paper as well as the FISH experiment in Melendez, et al. A new protocol was written to account for any adjustments made in the course of doing the protocol with the Gasch lab and for the first time at UW Madison.

Materials

• Stellaris FISH Probes (Biosearch Technologies)
• T₁₀E₁ buffer; 10 mM Tris-HCl, 1 mM EDTA, pH 8.0; (??)
• Yeast media (varies per experiment)
• Yeast strain(s) (varies per experiment)
• 50 mM Sodium Acetate, pH ~5
• 32% Paraformaldehyde (?)
• 70% Ethanol
• 1.2M Sorbitol (Sigma S6021)
• 1M Potassium dihydrogen phosphate KH₂PO₄ (Sigma P0662)
• 1M Dibasic potassium phosphate K2₂HPO₄ (Fisher BP363)
• β-Mercaptoethanol (Fisher O3446I)
• DAPI, dilactate (Sigma D9564)
• 200mM Vanadyl Ribonucleoside Complex (New England Biolabs S1402S)
• Lyticase (Sigma L5263)
• Phosphate Buffered Saline PBS 10X RNase Free (Ambion AM9932, or equivalent)
• Dextran Sulfate (Sigma D6001)
• 20X Nuclease-free Saline-Sodium Citrate (VWR 82021-484)
• Deionized formamide (Ambion AM9342, or equivalent)
• Nuclease-Free Water (Ambion AM9932, or equivalent)
• α-D-Glucose (Sigma 158968)
• 1M Tris-HCL, pH 8.0 (Ambion AM9855G, or equivalent)
• Glucose oxidase (Sigma G0543)
• Catalase (Sigma C3155)
• Poly-l-lysine 0.01% Solution (Sigma P8920)
• Prolong Gold Antifade Reagent (Invitrogen P36934)
• Eppendorf Tubes 2ml (??)
• Glass-bottom 96-well plates (Nunc 265300)

Solutions

Stocks

• FISH Probe stocks
• DAPI Stock Solution
• Vanadyl Ribonucleoside Complex (VRC) Stocks
• Lyticase Stocks
• 10% α-D-Glucose
• Glucose Oxidase stock

Buffers

• Buffer B
• Spheroplast Buffer
• Wash Buffer
• Hybridization Buffer
• Wash Buffer + DAPI
• GLOX without enxymes
• GLOX with enzymes

FISH Probes

Fish probes are designed using the probe designer from BioSearch technologies and ordered from BioSearch technologies.

Reconstituting the Dried Probe Stock

The probe stock should be sufficient to provide up to 200 or more hybridizations. For frequent use, aliquots of the probe stock can be stored at +2 to +8 °C in the dark for up to a month. Redissolve the dried oligonucleotide probe blend in 200 μL of T₁₀E₁ buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), to create a probe stock at a total oligo concentration of 25 μM. Mix well by vortexing and spin down. (Check with Leah if this is what she did to dissolve the probes and what final concentration they are at....Stellaris recommends 80uL for a final concentration of 12.5μM)

Short-Term Storage & Daily Use

Dissolved probe mix should be subjected to a minimum number of freeze-thaw cycles. For daily and short-term use, the mix can be stored at 4°C in the dark for up to a month.

Long-Term Storage

Store the probes in the dark at -20°C for storage lasting longer than a month.

DAPI Stock Solution

• DAPI, dilactate, catalog #D9564 from Sigma

To make a 5 mg/mL DAPI stock solution (10.9 mM for dilactate), dissolve the contents of one vial (10 mg) in 2 mL of deionized water (dH2O). Note: This DAPI derivative is not very soluble in phosphate-buffered saline (PBS).

Storage: For long-term storage the stock solution can be aliquoted and stored at ≤–20°C. For short term storage the solution can be kept at 2–6°C, protected from light. When handled properly, DAPI solutions are stable for at least six months.

Vanadyl Ribonucleoside Complex (VRC)

• New England Biolabs, S1402S
• Aliquoted and stored in the -20°C freezer. Stock solution is 200mM. Defrost the frozen stock from the freezer at room temperature. This can be reconsistuted to a green-black clear solution by incubating the tube at 65°C only if necessary.

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.

10% Glucose Solution

• α-D-Glucose
• Nuclease-free water

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.

Glucose oxidase stock

• Dilute glucose oxidase (Sigma, G0543) to 3.7 mg/mL in 50 mM sodium acetate, pH ~5

Buffer B

• 1.2M Sorbitol (approximate, see notes)
• 100mM KHPO₄ pH 7.5 (approximate, see notes)

To make mix together:

• 8 ml of 1M KH₂PO₄
• 41.5 ml of 1M K2₂HPO₄
• Sorbitol 109.3g

Spheroplast Buffer

• 890μL Buffer B
• 100μL VRC
• 10μL Lyticase (250U lyticase)
• 2μL β-Mercaptoethanol

Hybridization Buffer

• Dextran sulfate
• 20X saline-sodium citrate (SSC)
• Formamide (deionized)
• Nuclease-free water (to 10 mL final volume)

Mix together 1g of Dextran sulfate, 1ml of 20x 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

• 20X saline-sodium citrate (SSC)
• Formamide (deionized)
• Nuclease-free water (to 50mL total volume)

Mix together 5ml of SSC, 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 with DAPI

• DAPI stock at 5mg/mL
• 20X saline-sodium citrate (SSC)
• Formamide (deionized)
• Nuclease-free water (to 50 mL final volume)

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.

Anti-fade buffer WITHOUT enzymes

• 10% α-D-glucose in nuclease-free water
• 1 M Tris-HCl, pH 8.0
• 20X saline-sodium citrate (SSC)
• Nuclease-free water

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. (NOte: You will use 100µL of this buffer tto make "GLOX" buffer. Vortex and then transfer 100 µL of this 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 will be used as an equilibriation buffer.)

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

• 10% glucose in nuclease-free water
• 1 M Tris-HCl, pH 8.0
• 20X saline-sodium citrate (SSC)
• Nuclease-free water
• Glucose oxidase (3.7mg/mL)
• Catalase

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 buffer to another tube, to which one should add 1 µL of the glucose oxidase stock and 1 µL of mildly vortexed catalase suspension.

Protocol

Fixing Cells

1. Grow culture to Klett ~50-100 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 6.4% (4ml culture + 1ml paraformaldehyde). Incubate at room temperature for 45 minutes.
3. Spin the cells down at 3000 xg for 5 minutes. Resuspend in 1 ml Buffer B and transfer to an eppendorf.
4. Wash cells 3X with 1ml Buffer B by pipetting up and down gently. Spin down at 3000 xg 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 3000 xg (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).

Hybridization

1. Mix a small volume of the probe stock (1µL) with Hybridization Buffer 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. The working dilutions should be made into T₁₀E₁ (same as for the initial resuspension of the dried probe).
2. Gently aspirate the 70% ethanol off of the sample.
3. Add 1000μL of wash buffer with the same percentage formamide (10%) as the hybridization buffer and let stand for 2-5 minutes.
4. Spin 10 min at 2000rpm, aspirate the wash buffer and then add 100 µL of hybridization solution plus probe.
5. 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.
6. Add 1000μL of wash buffer to the sample. Pipette up and down gently.
7. Incubate at 37°C for 30 minutes in the dark.
8. Spin 10 min at 2000rpm, aspirate the wash buffer, then re-suspend in another 1 mL of wash buffer with 5 ng/mL DAPI for nuclear counterstaining.
9. Incubate at 37°C for 30 minutes in the dark.
10. Spin 10 min at 2000rpm, aspirate the wash buffer and resuspend in 2x SSC.
11. Spin 10 min at 2000rpm, aspirate the SSC and add the GLOX buffer without enzymes 1ml for equilibration; incubate for 1-2 minutes.
12. Spin 10 min at 2000rpm, aspirate the buffer and add 100 µL of GLOX buffer to which the enzymes (glucose oxidase and catalase) have been added.
13. Proceed to imaging

Image

1. Coat the 96 well optical plate with POLY-L-LYSIN solution, incubate at room temperature for 5 minutes, aspirate the solution, dry for 30 minutes in room.
2. Wash with water 3X and dry for 30 minutes in room.
3. Add cells in the GLOX buffer to the well and wait 30 minutes for the cells to settle down.( The amount of cells depends on the cell pellet you have before you add the GLOX buffer.)
4. Gently aspirate the buffer and wash with 1X PBS 3 times and then wash with 70% ETOH.
5. Cover the well with 50 µL of ProLong Gold Antifade Reagent proceeds to the scope.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

*Megan N McClean The Buffer B protocol is not exactly 1.2M sorbitol because the volume of the sorbitol added is not taken into account, similarly the KHPO₄ concentration is slightly less than 100mM. This is the protocol that the Botstein lab uses, so I'm assuming that it works fine.

*Megan N McClean This protocol is in the process or being optimized. Steps that may need to be modified (there may be more, this is just what I can think of at the moment):

• DAPI concentration

• Megan N McClean 17:27, 19 October 2011 (EDT): Some notes from Sandy in the Botstein lab:
  • He uses 10mls of about 3x10⁷== chemostat cells to start off with, spins those down and they end up in 1ml of 70% EtOH at the end of the first day, and then he takes 300μL of that to proceed to the hybridization steps. He does all of the hybridization steps in tubes, and does all of the washes with 1ml and then spins very gently (2000rpm on the Botstein small brown centrifuge). After our step 13 (was and resuspend in 2x SSC he does another spin down and resuspend and wash in 1x SSC.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

References

Botstein lab protocol: Botstein Lab FISH Protocol

Stellaris Protocols: Stellaris FISH Protocols

Contact

• Megan N McClean 14:01, 11 October 2011 (EDT)

or instead, discuss this protocol.