Product Information

• From what sort of cells do your metaphases come from for testing?
  Lymph Cells (cell lines)
• From which species of mouse are the mouse probes made?
  From about 6 different mouse strains: C57, C3H, SV129, BALB/c, PL/J, 101
• Is there any cross-hybridisation between mouse X and Y?
  Our lab is of the opinion that cross-hybridisation between mouse X and Y will be negligible. Some exists, but given the technical conditions, one is unlikely to see much.
• What is the concentration of the whole chromosome paint
  'ready to use' probe has 4ng/µl (600ng per 150µl vial). 'concentrated' probe has 20ng/µl (600ng per 30µl vial)
• What is the concentration of the chromosome-specific centromeric paint?
  'ready to use' chromosome-specific centromeric is 10ng/µl 'concentrated' chromosome-specific centromeric is 50ng/µl
• What is the size of the whole chromosome probes?
  The lab does not know this information. The size of the PCR amplified DNA used to make the probe is 500bp
• I want to use your probe with another from another company, will this work?
  Probes that have different Hybridisation mixes will not work well if mixed. It would require some experimentation on behalf of the user. The other option is to do the hybridisations consecutively
• What exactly is the probe detecting? Is it specific to a certain intron, the histones, or a common allele? Can you tell me the size of this probe in kb? Can you tell me the sequence?
  The whole chromosome paints DNA amplification is a random process and the labelling would cover a larger part of the chromosome DNA (approx 95%) but we cannot give you a sequence for it. We do not know what is the size of the whole chromosome probes. The size of the PCR amplified DNA used to make the probe is 500bp
• What is the DNA concentration of the probe and how much is required per test? (22x22 coverslip)
  There is approximately 700ng DNA per 1µl concentration, this can vary as the lab add as much as is required for a clear result. As for test requirement the protocol should be followed

Buffers

• What is in the Hybridisation Buffer?
  Whole Chromosome paints buffer: Proprietary
  Pan-centromeric probe buffer: 50% Formamide, 2x SSC, 0.02% Denhardts and 1% Dextran Sulphate
  Chromosome-specific centromeric probe buffer: 60% Formamide, 2x SSC, Denhardts, Salmon sperm DNA but no Dextran Sulphate
  Pan-telomeric paints buffer: 2xSSC, Denhardts, 30% Formamide

Centromeric / Telomeric Paints

• Do mouse pan-centromeric probes recognize the major satellite repeat elements or the minor satellite repeat elements in the mouse genome?
  The major satellite
• How much concentrated pan centromeric probe buffer should I use?
  On a 22x50 coverslip 2µl probe plus 25µl buffer, or on a 22x22 coverslip 1µl probe and 15µl buffer
• Are your pan-centromerics alpha-satellite probes?
  Our pan-centromeric probes consist of DNA alpha-satellite repeat sequences, generated by PCR
• Regarding the human pan telomeric probe, how many base pairs of the product and where specifically does the probe bind to in the telomeres? Tandomly repeated sequences, TAR or both?
  The telomere probe is a PCR product, so it's difficult to give size. It is generated using 2 primers that are 30bp with a 5x repeat telomeric sequence of a 6bp telo sequence. The repeat sequence is TTAGG
• Is it normal not to see any signal on the centromere using mouse whole chromosome paint?
  Yes. Competitor DNA is used to block the signal on the centromere. Because the same sequence occurs on all centromeres (hence the pan-centromeric probe) it's inclusion might lead to confusion
• Are the biotin labelled chromosome-specific centromerics less strong than FITC or Cy3?
  Yes, the direct labels give a better signal
• What do you recommend if I can't get Fixogum?
  Use clear nail varnish and a dry chamber, not a humidified one

Detections and Labels

• What is 4xT Solution?
  Detergent wash solution: 500ml 4x SSC and 250µl Detergent (1124-DT)
• I am a first time user, which label do you recommend?
  We recommend Biotin. Over- or under-denaturation of our FITC paints results in no signal
• Counterstain 2 is not mentioned in the new protocol for 1089-KB kit, Why is that?
  Counterstain 2 (Propidium Iodide) has a red signal, which could be confusing if used with Texas Red ot Cy3 probe. Follow the new protocol just using counterstain 1 (DAPI) and you'll be fine. However, if you are using FITC, then you can continue to use it, in the same quantities as counterstain 1
• Can I perform ratio labelling with your paints?
  Yes, we recommend you use our concentrated paints, and begin with equal quantities of each label. The user should determine the required ratio by experimentation
• Can FITC paints be used as direct-labelled probes?
  Our centromeric and pan-telomeric FITC paints can certainly be used as direct-labelled paints. However, although our whole chromosome paints have been used successfully thus, we would recommend a round of amplification to ensure best results
• Can I use a PI filter Cy3?
  Yes, or Rhodamine
• I would like to do triple painting with biotin, cy3 and FITC. However, I'm worried that when I detect biotin, it might affect the signals of the FITC and Cy3.
  If you use Cy5-avidin to detect the biotin, it should not affect the Cy3 and FITC signals. Cy5-avidin can be used together with either layer of antibodies (F1 and F2) used to detect the FITC
• Can human whole choromosome paints be used in immunolabelling of proteins?
  No
• Which paint is best used to distinguish between human and mouse cells?
  We recommend our Star*FISH Human Chromosome-specific 22 paint. This will pick out chromosome 22, which mice do not have. Alternatively, human and mouse centromerics should not cross-react, due to the divergence between species of the repetitive sequences. Otherwise, mouse and human Y probes do not cross react (you have to increase the hybridisation time from overnight to about 3 days to get cross-reactivity). However, if you're not always using male cells, then this option is not suitable.
• When carrying out washing steps prior to detecting paints, can I use the same coplin jar of detergent wash solution?
  No, you should use a fresh detergent wash solution each time. This is important step; if the washing is not performed properly, you will get lots of background

Bovine Paints

• What labels are used in the bovine X/Y probe?
  The X is labelled with Biotin , and the Y with Cy3
• What is the average fragment size in the bovine paints?
  Between 200-500bp
• Concerning the bovine X/Y probe: If the X is labelled with biotin, won't an avidin-FITC label provide suffient signal? Why the additional anti-avidin-FITC?
  yes, X can be detected with a single layer especially in an interphase cell. Two layers gives a brighter signal and were used for FISH in sperm
• What magnification do you use for the bovine XY probe on sperm?
  40 or 63
• Is the bovine sperm intact at the end of the FISH experiment?
  Yes, although the tails are lost
• Will your bovine probes work on the PAR (pseudo autosomal region) in cattle?
  The probes are not specific for a region as they are for the whole chromosome and would not work on specific gene
• My bovine sperm cells seems to be damaged by the pepsin treatment step, and possibly lysed during denaturation. What should I do?
  Reduce the concentration of the decondensation solution but keep the timing the same

Rat Paints

• Can I use mouse paints on rats?
  No, the chromosomes do not correspond betwen species. Even mouse pan-centromerics do not work on rats
• Does rat 12/Y contain Cot1?
  No
• Why is rat 12/Y dual labelled?
  The Y is Cy3 (red) and the 12 is FITC (green). However, the centromere and short arm of 12 contains the sequence that is homologous to Y, so often looks yellow/orange where the labels combine. Because the staining of the Y is so strong, it usually over rides the staining of the homologous region on Y, making Y appear all red There are also areas of cross-hybridisation with chromosome 3 (will be yellow/orange) and other smaller chromosomes, which may appear as little spots
• Why is 12 included with Y in the rat 12/Y paint?
  The 12 was left in the probe so that all cross-hybridised areas appear yellow/orange (except on Y, as explained above) This therefore differentiates Y from the cross-hybridised regions. Thus if anything stragn is going on with Y (translocations etc) then one knows it *is* Y, not one of the areas of cross-hybridisation
• Can the rat 12/Y probe be used to reactive with inbred Lewis rats?
  Yes
• Can the probe be used on FFPE tissues? (formalin-fixed, paraffin embedded)
  Although this has never been tried in our lab, we have customers that have used them and received good results

Dog Paints

• Is cross-hybridisation normal in the dog paints?
  Yes - unfortunately this is not one of our best! There are significant cross-hybridisation with heterochromatin and centromerics

PRINS

• May PRINS be adapted to a probe of the customers own design? For example, to label very small portions of the gene of interest in order to identify an integration site
  It would be best to do a chromosome painting first and then use the PRINS

Contig 21

• Now that contig 21 has been discontinued, what can I use?
  The Down's Syndrome specific regions are 21q22.2-21q22.3 and 21q22.1-21q22.2 and 21q11.12-21q22.12. Use the ALTech probe that encompasses this region and you should be fine. You can also visit http://wehih.wehi.edu.au/gdbreports/Chr.21.omim.html

Methods and Protocols

• How critical is the aging step?
  The aging step is important, this is why the method states to drop the slides one day and hybridise the following. If not heating at 65°C for one hour would "age" and allow you to do both in the same day
• Would a longer hybridisation time (48 hours), lead to more background?
  Yes, the longer the hybridisation the stronger the background
• Does Cambio have a good resource for metaphase slide preparation?
  Yes, the protocols booklet to be used with our products
• I am pepsin-treating my slides. Will this affect the denaturation conditions?
  Yes, you should reduce the denaturation time from 1.5-2 min to 1 min, keeping the temperature the same.
• I need to clean up my slide, and I'm already doing pepsin treatment. How much blocking protein should I add, and when?
  Blocking protein is put into the paints anytime before denaturing of the paints, at 1µl per test
• Can I stain more than one chromosome on one slide?
  If you plan to use more than one paint on a slide, or experiment with ratio labelling, we strongly recommend the use of our concentrated paints. These are supplied with a separate vial of hybridisation buffer, giving you control over the volume of probe and buffer on each slide. We suggest you start with a 1:1 ratio, and use the multiple detection protocols (see brochure) as appropriate
• Can I use whole chromosome paints and pan-centromeric paints together?
  Yes. You can either do it in two stages, or all at once.
  Two stage technique:
  
  1. Apply the whole chromosome paint first, then, use these washes the next day:
     • Wash in 2x SSC at 42°C for 5 min to remove cover slips
     • Wash in 50% formamide in 0.6% SSC at 42°C for 5 min. Repeat
     • Wash in 2x SSC at 42°C for 5 min. Repeat.
  2. After the washes, use protocol G to apply the pan centromeric paint and use the appropriate detection protocols for your labels.
  
  One stage technique:
  1. Add centromere probe (denatured) in a small amount of hyb mix to the Reannealed whole chromosome paints just before adding to slide. The centromere probes do not need reannealing as the paints do, so they have to be denatured separately. Suggested amounts: 2ul/slide + 5ul for denaturing centromere probe
• Can I use Mouse probes and human probes together in the same experiment?
  There is some possibility of cross-hybridisation, but to achieve this you would need to hybridise for about 3 days, so this is possible in experienced hands
• What can we use instead of Cow Gum?
  Qbiogene's Fixogum is recommended by the lab. Second best is rubber solution from your local bicycle repair shop!
• I've got half way through my experiment and realised I have run out of one of my detection reagents. Is my experiment ruined?
  Not necessarily. Store your slide at -20°C until you have all the reagents you need. If you have counterstained it, you will need to wash off the counterstain:
  At room temperature wash in 70% ethanol briefly (max 2 min), wash in 4XSSC for 5 min, then perform detection
• Can I use Star*FISH paints in interphase?
  Although our paints will give a signal in interphase, there are a number of potential problems which should be borne in mind. Primarily, because the DNA is no longer super-coiled, the signal will be less intense. This will not be the case with centromeres. However, there is also the danger that the area under investigation might be concealed beneath another chromosome. Therefore, we do not recommend the use of our paints in interphase.
• Is there any way I can get round the problem of acrocentric cross-hybridisation?
  Yes! Use American Laboratory Technologies' arm paints. Unlike flow-sorted chromosome DNA FISH probes, these microdissected 13q, 14q, 15q, 21q and 22q DNA FISH probes evenly cover the entire length of the q arm of those chromosomes, and show no cross-hybridisation to other acrocentric chromosomes either centromere region or p arms
• I want to use your probe with another probe from another company will this work?
  A Probes that have different hyb mixes will not work very well if mixed. It would require some experimentation on behalf of the user. The other option is to do the hybridisations consecutively
• Can I denature that probe and the slide together?
  We don't recommend this as it always produces more background.
• After Hybridization with your probe, Could I use avidin-Biotin enzymatic system (ABC) to develop the signal? Let avidin-biotin-conjugated-HRP to bind the biotin of Y-Probe then, allow the substrate DAB to generate the colour?
  You can use a different detection system as long as you have an avidin-biotin attaching to the probe (labelled with Biotin). The lab has never tried this method so we would not be able to help on the detection steps
• Can I carry out FISH detection and immunostain on the same slide?
  These 2 links imply you can, but we don't actually know if it works with our chromosome paints. http://biochemistry.ucsf.edu/~panning/protocols/protocol1.html and http://info.med.yale.edu/genetics/ward/tavi/fi21.html
• Can I use your mouse or human paints of sperm? Should I follow the bovine sperm protocol?
  Yes, you can use the mouse and human paints on sperm, but you should NOT use the bovine protocol. Bovine sperm is much more tightly condensed than mouse or human, so requires different preparation. A protocol is available in W A Robbins et al, American Journal of Human Genetics, 1993 vol 52 p799