- How should my slides (with metaphase preparations) look prior to doing FISH?
- After FISH, I have a dim signal and a "haze" covering the slide. How can I correct this?
- Why is the counterstain too faint?
- Why is the cell morphology of my sample distorted?
- How long can I store my prepared slides and still hybridize them effectively?
- After hybridization, how long can I store my slides and still see fluorescence?
- Can I perform FISH after G banding my metaphase spreads?
How should my slides (with metaphase preparations) look prior to doing FISH?
Evaluate your slide preparations under a phase contrast microscope. Good preparation: Chromosomes and nuclei should be dull gray, nonrefractile, and with minimal cytoplasm. Poor preparation: Cells will be "phase bright", appearing white with a halo around the nuclei. Possible cause: Sample on the slides may have dried too quickly during preparation. Solution: - Increase the humidity around the slide when "dropping" the sample byplacing it on a rack over a 65 C water bath for 20 minutes. - Do not bake the slides; baking slides can adversely affect the hybridization results. - Dry slides at room temperature overnight. Best results are obtained when the slides are aged overnight at room temperature. - Slides prepared and used on the same day are less than optimal, butthe following steps can be used to prepare slides in one day: dry theslide first on a 45 C slide warmer for 30 minutes, then dehydrate theslide for one minute each in 70%, 85%, and 100% ethanol solutions priorto the denaturation of the target DNA (step 1 for FISH).
After FISH, I have a dim signal and a "haze" covering the slide. How can I correct this?
Usually, the cause is from too high a density of cells dropped onto the slide.The haze is from the cell cytoplasm. Try washing the cell pellet with fresh fixative, dilute the sample and drop onto a new slide. Some types of samples (touch preps, smears of bone marrow or blood,paraffin-embedded samples) may be more prone to a "haze" problem. These types of samples may require additional treatments: Smear: 30 second wash in 70% acetic acid, air dry, 30 second wash in 100% methanol. Paraffin-embedded: increase protein digestion time.
Why is the counterstain too faint?
The slides may not have been dried properly before the counterstain wasapplied. Be sure to age the slides for 24 hours after dropping the cells. Try dehydrating the samples by a series of ethanol washes, then re-counterstain. Also, be sure you have the proper filter for viewing the counterstain. There are two concentrations of DAPI counterstain available from Vysis. DAPI II (125 ng/mL) is for use with CEP® and LSI® probes is an eight-fold dilution of DAPI I (1000 ng/mL) which is for use with WCP® Chromosome Paints. Use of DAPI II with WCP probes may produce too faint a counterstain.
Why is the cell morphology of my sample distorted?
Different tissue or sample types may require different denaturation times. If the denaturation time is too long for the sample type, the cells will appear distorted. Try a four minute denaturation time to start and then adjust accordingly.
After hybridization, how long can I store my slides and still see fluorescence?
If the slides are stored at -20 C and not exposed too frequently to the high intensity UV light of the mecury lamp, then they will last several months. Signals will fade over time and with light exposure.
Can I perform FISH after G banding my metaphase spreads?
Yes, the following reference contains a procedure:
JalalSM, Law ME, Christensen ER, Spurbeck JL, Dewald GW. Method forsequential staining of GTLbanded metaphases with fluorescentlabeledchromosome specific paint probes. Am J Med Genet 46:98103, 1993.