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 All chromosome slides that I have prepared have a halo around the chromosomes or cells. What could have caused such phenomenon?
After fixing the cell suspension and placing droplets on the slide, insufficient drying time will result in a halo around the chromosomes or cells. We recommend that you increase the humidity of droplet environment. For example, place the blank slides over a 65°C water bath and prepare the slides after 20 minutes of vaporization.
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What kind(s) of chromosome FISH slide is most suitable for the FISH experiment?
The best chromosome slide is one with evenly spread out chromosomes when examined under the microscope. Chromosomes should not overlap and intertwine, and have a dark gray or close to black color.
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How important is pre-treating of paraffin-embedded slides?
It is critical to deparaffinize and digest the sample slides with enzyme; it allows access of probe DNA to the cell nucleus and facilitates probe binding to the target regions.
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If the coverslip and the slide are not completely sealed by the rubber cement, will it affect the experiment result?
To avoid excessive evaporation of probe during hybridization, which may affect the experiment result or even result in dirty background, please make sure the coverslip and the slide are sealed off completely with rubber cement.
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After placing probe on the sample, how do I prevent bubbles from forming when placing the coverslip over the slide?
We recommend that you tilt the coverslip at a 45 degree angle when placing it onto the slide.
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During FISH hybridization, how should I place the slides in 37°C oven or incubator?
We recommend that you place the slides in a crisper container. Place a layer of wet paper towel on the bottom to maintain the humidity in the container.
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How long should hybridization be performed?
Hybridization should be performed for 12-16 hours. Shorter hybridization times tend to reduce signal intensity, while longer times more likely lead to increased background noise.
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How long can the denature solution be used?
The denaturing solution can be used for up to a week if it is kept properly at 4°C.
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What can happen if the sample slides are under- or over-denatured?
Denaturing over time or at a temperature higher than recommended may cause over-denaturing, which causes a loss of chromosomal DNA, eventually leading to distorted chromosome morphology and weak FISH signals. In contrast, if denaturation is inadequate, the signals may be weak because the probes cannot hybridize to the target complementary DNA.
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What are the most critical factors for denaturing?
It is critical to denature the chromosomes adequately prior to FISH. Temperature and duration are the two most important factors for denaturing. It is recommended to equilibrate the denaturing solution in 72°C water bath for more than 30 minutes before use.
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