Fluorescence in situ Hybridization Protocol (FISH for Yeast)

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 23 ^o C for 3 hours. Go to step 3
   c) Temperature sensitive mutants: transfer cells to 37℃ 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℃.
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℃).
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℃ 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℃ 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℃). Incubate 1 hour at 37℃.
   
   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℃) 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℃ bath (see step 16)
   
   .Denaturation:
7. Prewarm 60 ml denaturing solution (70% formamide, 2X SSC) to 70-72℃ in a coplin jar.
8. Place slides on 37℃ 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℃ drop. Allow denaturing solution to reheat to 70℃ before denaturing additional slides.
10. Immediately immerse slides through a sequence of cold (-20℃) 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℃) and incubate for 15 minutes at 37℃. 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℃) 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℃. 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℃ 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℃.