Koshland Web Site/Methods
FLUORESCENCE IN SITU HYBRIDIZATION PROTOCOL
Modified 9/96
CELL PREPARATION: (Day 1)
  1. Grow cells in YEPD to early to midlog phase (O.D.600 of 0.3-0.4 for haploids and 0.5-0.6 for diploids).
  2. a) Asynchronous cells: proceed to step 3.
    b) Nocodazole blocked cells: add nocodazole to a final concentration of 15 ug/ml then incubate cells at 23oC for 3 hours. Go to step 3
    c) Temperature sensitive mutants: transfer cells to 37°C and incubate 3 hours. Go to step 3
  3. Fix cells by adding 100 ul 36% formaldehyde to 1 ml of cells and incubate for 2 hours at 23°C.
  4. Transfer 1 ml fixed cells to an eppendorf tube and pellet cells 20 seconds at 10 K. Resuspend cells in 1 ml distilled H2O then pellet cells 20 seconds at 10 K. Wash cells 2X more using 1 ml H2O per wash. Resuspend cells in 500 ul spheroplast buffer (At this point cells can be stored overnight at 4°C).
  5. Polylysine coat slides by adding 10 ul polylysine solution (1 mg/ml in H2O) to each well on slide. Incubate at room temperature for 10 minutes. Remove the polylysine then wash 2X with H2O and allow to air dry.
  6. Spheroplast cells by adding 10 ul beta-mercaptoethanol (1/50 cell vol), then add 5 ul (1/100 cell vol) of 3 mg/ml Zymolyase T100 (ICN or seikagiuchi zymolyase). Incubate cells for 1 hour at 23°C in H2O bath (no shaking).
  7. Pellet cells for 5 seconds at 10 K and resuspend gently (using pipetman) in 1/2X spheroplast buffer (use a volume equal to the spheroplast volume).
  8. Add 10 ul of cells to each well. Incubate for 10 minutes at room temperature.
  9. Remove the liquid in each well using a pipetman.
  10. Slowly add 20 ul 0.5% SDS to each well and incubate for 10 minutes at room temperature.
  11. Remove SDS using pipetman (hold pipetman perpendicular in the center of the well). Allow wells to air dry (takes about 5 minutes).
  12. Place slides in a coplin jar containing 3:1 methanol:acetic acid (freshly made). Incubate for 5 minutes at room temperature.
  13. Remove slides and place them on a paper towel to and allow to air dry overnight at room temperature. (Note: I often air dry for several days until slides no longer emit an odor of acetic acid).
  14. Slides can be stored for at least 6 months at 4°C in vacuum desiccator.

IN SITU HYBRIDIZATION: (Day 2)

RNase treatment:

  1. Dilute RNase A stock to 100 ug/ml using 2X SSC then add 10 ul to each well.
  2. Place slides into humid chamber (prewarmed to 37°C). Incubate 1 hour at 37°C.

    Dehydration:
  3. Remove slides from humid chamber and place slides in a coplin jar containing 2X SSC (room temperature). Incubate 2 minutes at room temperature (agitate slides after one minute).
  4. Wash slides 3X more (2 minutes/wash) using fresh 2X SSC. (For these washes, we prepare three coplin jars each containing 50 ml 2X SSC [room temp] and transfer slides to a new coplin jar for each wash).
  5. Place slides through a series of cold (-20°C) ethanol washes in coplin jars (2 minutes/wash). The first wash is 70% ethanol, followed by 80% then 95% ethanol washes. Use fresh 70% EtOH solutions in all steps; all other ethanol solutions can be reused through the remainder of the protocol.
  6. Allow slides to air dry at room temperature. (slides can be stored overnight). Note: Place an aliquot of 50% dextran sulfate in a 70-72°C bath (see step 16)

    .Denaturation:
  7. Prewarm 60 ml denaturing solution (70% formamide, 2X SSC) to 70-72°C in a coplin jar.
  8. Place slides on 37°C slide warmer for 5 minutes.
  9. Incubate slides for 2 minutes in the preheated (70-72oC) denaturing solution (agitate slides periodically during denaturation). Don't heat more than three slides simultaneously in one coplin jar because each room temperature slide causes about a 1°C drop. Allow denaturing solution to reheat to 70°C before denaturing additional slides.
  10. Immediately immerse slides through a sequence of cold (-20°C) ethanol washes in coplin jars (1 minute/wash). The first wash is 70% ethanol, followed by 80%, 90% then 100% ethanol washes.
  11. Allow slides to air dry at room temperature.

    Proteinase K treatment:
  12. Dilute stock proteinase K to 200 ug/ml using 20 mM Tris HCl pH 7.8, 2 mM CaCl2 then add 10 ul to each well.
  13. Place slides into a humid chamber (prewarmed to 37°C) and incubate for 15 minutes at 37°C. During this incubation prepare probe in hybridization mix as described in step 16 below
  14. Remove slides from the humid chamber and immediately immerse slides through a series of cold (_20°C) ethanol washes as described in step 10.
  15. Allow slides to air dry at room temperature.

    Probe hybridization:
  16. Prepare probe DNA in hybridization solution as follows; Make a stock hybridization mix by adding 250 ul formamide, 100 ul 10X SSCP, 100 ul 50% dextran sulfate and 20 ul 10 mg/ml sonicated salmon sperm DNA. Warm the hybridization mix to 70°C. For each pair of wells to be hybridized add 9.4 ul hybridization mix to 1 ul probe DNA and mix thoroughly (see helpful hints section for hybridization buffer preparation and probe DNA concentration).
  17. Denature probe/hybridization mix by incubating 10 minutes at 70°C then quick chill in an ice-water bath.
  18. Add 5 ul probe/hybridization mix to each well (I use two wells for each probe)
  19. Cut a piece of parafilm so that it is slightly larger than 2 wells. Use a forceps to cover both wells with the parafilm (make sure no bubbles are present). Fill a 1 cc syringe with rubber cement and place a layer of rubber cement around the edges of the parafilm. This seals in the probe and prevents mixing with other probes. Repeat for each set of 2 wells (ie probes).
  20. Incubate slides overnight (at least 16 hours) in a humid chamber at 35°C.

IN SITU HYBRIDIZATION: (Day 3)

Post-hybridization wash:

  1. Prewarm post-wash solution (60% formamide, 2X SSC) to 37°C in a coplin jar. In addition, prewarm 100 ml of 2X SSC to 37°C (50 ml in each of two coplin jars).
  2. Remove slides from humid chamber, peel off coverslips and incubate slides 20 minutes in the 37°C post-wash solution (agitate slides every 5 minutes).
  3. Transfer slides to the first coplin jar containing 2X SSC. Incubate slides for 5 minutes at 37°C (agitate slides after 2 minutes). Transfer slides to the second SSC containing coplin jar and incubate as described for the first SSC coplin jar.
  4. Transfer slides to a coplin jar containing room temperature 1X PBD (phosphate buffered detergent). Incubate for 2 minutes (agitate slides after 1 minute).
    DO NOT ALLOW WELLS TO DRY AT ANY TIME PAST THIS POINT!!!!!!!!!!!!!!!!!!

    Detection: This protocol can be used to detect either digoxigenin or biotinylated DNA probes. We use digoxigenin probes, primarily because it is much cheaper.
    a) Digoxigenin probes: Detection by i) mouse anti-DIG antibody, ii) FITC conjugated goat anti-mouse antibody and iii) FITC conjugated pig anti-goat antibody. All antibodies are diluted 1:250 in 10% horse serum just prior to use.
    b)Biotinylated probes: Detection is via FITC-avidin and anti-avidin antibodies (from the ONCOR in situ hybridization kit).
    Note: preparation of digoxigenin or biotin labelled probes is described in equipment and solutions section.
  5. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off liquid near slide edges. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul Mouse anti-DIG antibody to each well. Incubate 20 minutes at 37°C in a humid chamber (prewarmed to 37°C). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 1 from ONCOR kit instead of horse serum then add 7.5 ul FITC-avidin to each well instead of mouse antibody.

    Amplification:
  6. Remove slides from humid chamber and incubate slides for 2 minutes in a coplin jar containing 1X PBD (room temperature). Agitate slides after one minute. Repeat wash 2X in fresh 1X PBD at room temperature for 2 minutes/wash.
  7. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off excess liquid near wells. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul FITC conjugated goat anti-mouse antibody to each well. Incubate 20 minutes at 37°C in a humid chamber (prewarmed to 37°C). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 2 from ONCOR kit instead of horse serum then add 7.5 ul anti-avidin antibodies to each well instead of goat antibody.
  8. Repeat PBD washes as described in step 26.
  9. Remove one slide at a time from 1X PBD, quickly tilt slide to drain off excess liquid and blot off excess liquid near wells. Digoxigenin probes: Immediately add 7.5 ul 10% horse serum to each well containing a hybridized probe. Incubate 5 minutes at room temperature in a humid chamber. Add 7.5 ul FITC conjugated pig anti-goat antibody to each well. Incubate 20 minutes at 37°C in a humid chamber (prewarmed to 37°C). Biotinylated probes: follow digoxigenin probe instructions except use 7.5 ul blocking reagent 1 from ONCOR kit instead of horse serum then add 7.5 ul FITC-avidin instead of pig antibody.

    Chromosome staining:
  10. Repeat PBD washes as described in step 26.
  11. Remove one slide at a time from 1X PBD, tilt slide to drain off excess liquid and blot off excess liquid near wells.
  12. Add 5 ul propidium iodide/antifade solution to each well (warm to room temperature before adding since this solution is very viscous at -20°C). Cover with glass coverslip. Remove any bubbles by pressing coverslip gently with the eraser end of a pencil.
  13. Fill a 1 cc syringe with rubber cement and seal the coverslip to the slide by adding rubber cement along the coverslip edges.
  14. View using fluorescence microscope. (store slides at -20°C).

HELPFUL HINTS:

  1. The most important factor is the making of the digoxigenin or biotinylated probes. If the probes are not good then everything else is meaningless.
  2. The concentration of labelled probe DNA used for hybridization must be determined
    empirically. I start with 0.5 ul probe stock solution + 0.5 ul H2O in 9.4 ul hybridization buffer (2 fold dilution; approximately 2 ng probe DNA) and test a series of 3 fold dilutions. Typically, I can dilute the stock labelled probe 15 to 30 fold.
  3. To make hybridization buffer; a) heat an aliquot of 50% dextran sulfate to 70°C to make the solution less viscous. I heat the dextran sulfate during RNAse treatment of slides.; b) make a large volume of hybridization solution by adding 250 ul formamide, 100 ul 10X SSCP and 20 ul sonicated salmon sperm DNA [10 mg/ml] and mix using a P1000 pipetman. Add 100 ul of the prewarmed 50% dextran sulfate. [Even the 70°C dextran sulfate is viscous so I use graduated pipet tips which have a 100 ul markings and bring the volume of dextran sulfate to the 100 ul mark; alternatively, cut off the tip of the P200 tips]. Mix well using a P1000 pipetman; c) place the hybridization mix at 70°C until ready to add probe. I do this before the proteinase K treatment of slides.; Discard any unused 50% dextran sulfate that has been heated to 70°C. I only heat it once since the dextran sulfate doesn’t appear to work as well after several reheatings; d) add probe DNA to hybridization mix as described in step 2 above and denature 10 min at 70°C to 72°C (I denature probe during the time of proteinase K treatment of slides).
  4. The temperatures of incubator and solutions should be monitored closely. The temperature of the incubator used for overnight hybridization of probe should be no higher than 36°C. I try to get the incubator to be at 35°C. When our incubator temperature drifted up to near 40°C we detected little or no probe hybridization. The denaturation temperature should be 70 to 72°C.
  5. Cells used for this protocol should be fresh. We like to either inoculate cells from a fresh saturated culture into liquid then grow overnight for processing the next day. Alternatively, if stock cells have been stored as saturated cultures for several weeks, we inoculate cells into fresh liquid, grow overnight. The next day mid or late log cells are inoculated into fresh liquid and grown overnight for processing the following day.
  6. Cells must be well spheroplasted for best results. During spheroplasting, cells become dark but after approximately 1 hour the cytoplasm becomes more transparent and you may see the nucleus as a darker staining sphere. We use 20 mM phosphate in our spheroplast buffer since this works better than 100 mM. Finally, try to keep cell concentration at the specified level by diluting cells to the proper OD 600 equivalence before starting spheroplasting.
  7. Slides can be stored after RNase treatment. However, once slides are denatured proceed through the protocol until probes have been added to slides and hybridization has begun.
  8. The 36% formaldehyde stock bottle used in our lab is kept at room temperature after it has been opened. If your lab immediately freezes aliquots after opening the bottle, it is possible that you are over fixing relative to our lab. Try performing a shorter fix, perhaps only 1 hr to 1 1/2 hr.
  9. If you still are having problems, try performing a second denaturation step after the proteinase K treatment. This should increase the FISH signal but can also increase background. This has made it difficult to see a good FISH signal for rDNA. It might be that simply diluting the probe more will reduce the higher background. Alternatively, try diluting the anitbodies 1:500 instead of 1:250.

SOLUTIONS and EQUIPMENT

DAY 1 SOLUTIONS

SPHEROPLAST BUFFER
1M Sorbitol, 20 mM KPO4 pH 7.4
(formula for 50 ml below)
25 ml 2 M Sorbitol
0.83 ml 1 M K2HPO4
0.17 ml 1 M KH2PO4
24 ml H2O
 ZYMOLYASE STOCK SOLUTION. Make 3 mg/ml zymolyase T100 (ICN or seikaguchi zymolyase) in 10% glucose (Store 25 ul aliquots at -20°C)
POLYLYSINE SOLUTION. 1 mg/ml polylysine in H2O. (Store at 4°C or -20°C) 1% (V/V) TRITON-X100
0.5% (W/V) SDS (ULTRAPURE) 3:1 METHANOL:ACETIC ACID (make fresh)

DAY 2 SOLUTIONS

PROTEINASE K STOCK. Stock is liquid proteinase K from Boehringer Mannheim, catalog # 13330224-04. The stock concentration varies from batch to batch (10-15 mg/ml). Concentrated stock is stored at 4°C. Just prior to use, stock is diluted to 200 ug/ml using 20 mM Tris-HCl pH 7.8, 2mM CaCl2. RNASE STOCK SOLUTION. 10 mg/ml RNase A is made DNAse free as described in Maniatis. Stock is stored in 1 ml aliquots at -20°C
FORMAMIDE (triple distilled): ONCOR formamide (Catalog # S4117)
ONCOR, 209 Perry Parkway, Box 870, Gaithersburg, Maryland 20877		 DENATURING SOLUTION
Final conc. Recipe for 60 ml
70% Formamide 42 ml Formamide
2X SSC 6 ml 20X SSC
12 ml H2O
20 X SSC
Final conc. Recipe for 1 liter
3.0 M NaCl 175.3 g NaCl
0.3 M Na Citrate 88.2 g
800 ml H2O
pH to 7.0 using NaOH then bring to 1000 ml using H2O		 10 X SSCP
Final conc. Recipe for 100 ml
1.5 M NaCl 8.75 g NaCl
0.15M Na Citrate 4.41 g
0.2 M NaH2PO4 2.4 g NaH2PO4

pH to 6.0 using NaOH
HYBRIDIZATION SOLUTION
Final conc. Recipe for 10.4 ul
probe DNA 1ul probe DNA (approp. conc.)
50% Formamide 5 ul Formamide
10% Dex, Sulfate 2 ul 50% Dextran Sulfate
2X SSCP 2 ul 10X SSCP
400 ug/ml salmon 0.4 ul 10 mg/ml sonicated
sperm DNA salmon sperm DNA

DAY 3 SOLUTIONS

POST-WASH SOLUTION
Final conc. Recipe for 60 ml
60% Formamide 36 ml Formamide
2X SSC 6 ml 20X SSC
18 ml H2O		 1X PBD
0.1 M NaH2PO4
0.1 M Na2HPO4
0.1% (w/v) Nonadet P40
ANTIFADE/PROPIDIUM IODIDE
Dissolve 50 mg p-phenylenediamine (Sigma) in 5 ml 1X PBS, adjust to pH 9.0
Add 45 ml glycerol & stir to homogeneity.
We store this in 1 ml aliquots at -80 in the dark. When using for FISH we add 5µl of propidium iodide (2.5mg/ ml stock solution in H2O) to 1 ml and keep this
dark at -20.

SLIDES. 10 well slides with 5 mm diameter circular wells (autoclavable) obtained from Roboz Surgical Instrument Co. Inc., 9210 Corporate Blvd., Suite 220, Rockville, Maryland 20850).
HUMID CHAMBER. Layer the sides of a rectangular tupperware dish or large petri dish with wet paper towels. Place parallel strips of tygon tubing or glass rods on the bottom of the dish (to serve as a platform for the slides). Cover the container with the lid to seal the humid chamber. DETECTION OF DIGOXIGENIN LABELLED PROBES
ANTIBODIES: 1) Mouse anti-digoxigenin IgG1 (monoclonal) obtained from Boeheringer Manheim Biochemicals (catalog # 1333 062). 2) Goat anti-mouse IgG (H+L) fluorescein (FITC) conjugated (affinity purified) obtained from Jackson Immunoresearch Laboratories catalog # 115-095-003 (phone 1-800-367-5296). and 3) Swine anti-goat IgG fluorescein conjugated (affinity purified) obtained from Boeheringer Manheim Biochemicals (catalog # 605270). Just prior to use, all three antibodies are diluted 1 to 250 using 10% horse serum
10% HORSE SERUM: 100% horse serum (heat inactivated) was obtained from Gibco BRL (catalog # 230-6050AG) and filter sterilized. The working blocking reagent is 10% horse serum in 1X TBS (with 0.02% sodium azide). The formula for one liter of 10X TBS is; 80g NaCl, 2g KCl, 30g Tris base dissolved in 800 ml H20 then pH adjusted to 7.4 using 1M HCl. Bring to 1 liter using H20 and autoclave DETECTION OF BIOTINYLATED DNA PROBES
Use Chromosome In Situ Kit; Signal Amplification Reagent Set (Catalog # S1352-SET) from ONCOR, 209 Perry Parkway, Box 870, Gaithersburg, Maryland 20877
PREPARATION of DNA for MAKING PROBES
1) Digest CsCl purified cosmids with Sau3A
2) Phenol/CHCl3 extract 1X, then CHCl3 extract 1X
3) EtOH ppt (add 1/10 vol 5M NaCl, 2 vol 100% EtOH)
4) Resuspend in 1X TE pH 8.0		 DIGOXIGENIN LABELLED DNA PROBES. The enzymes for nick translation were obtained from the BioNickTM Labelling System from GIBCO BRL (Catalog # 8247SA). The protocol is similar to that described for biotinylation except that 1 ul Digoxigenin DNA labelling mixture (Boehringer Mannheim Biochemicals; catalog # 1277 065) and 5ul 10 X nick translation buffer (0.5 M Tris-HCl pH 7.8, 50 mM MgCl, 100 mM beta-mercaptoethanol, 100ug/ml BSA) are substituted for 5ul 10 X dNTP mix from the BioNickTM Labelling kit. Note: The BMB Digoxigenin DNA labelling mixture contains all the required nucleotides including digoxigenin-UTP. It is listed as 10X but I use it as a 50X solution for nick translation.
BIOTIN LABELLED DNA PROBES. Use BioNickTM Labelling System from GIBCO BRL (Catalog # 8247SA)
a) Set up 3 tubes of Sau3A digested DNA; tube A 300 ng DNA, tube B 100 ng DNA and tube C 30 ng DNA
b) Add reagents from BioNickTM kit to each tube as described in kit
NOTE: we use less DNA than the kit suggests but add reagent volumes as stated for 50 ul reaction using 1ug DNA c) Incubate for 1 hr at 16°C
d) Add 5 ul 10X stop buffer
e) Precipitate DNA by adding 5 ul 10 mg/ml sonicated salmon sperm DNA, 6 ul 3M Sodium Acetate and 120 ul cold 100% EtOH.
f) Incubate 2hr at -20°C.
g) Pellet DNA by microfuging 15 minutes and discard supernatent
h) Resupend in 100 ul H20.
i) Reprecipitate DNA by adding 10 ul 3M Sodium Acetate and 200 ul cold 100% EtOH. Incubate 2hr at -20°C.
j) Pellet DNA by microfuging 15 minutes and discard supernatent.
k) Wash pellet with 1 ml cold (-20°C) 70% EtOH. Vacuum dry pellet.
l) Resuspend DNA in 25 ul 1X TE pH 8
Comments:
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