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℃ 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℃ 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℃ 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℃. 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℃ to 72℃ (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℃. I try to get the incubator to be at 35℃. When our incubator temperature drifted up to near 40℃ we detected little or no probe hybridization. The denaturation temperature should be 70 to 72℃.
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.

DAY 1 SOLUTIONS

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