i prepare slides with typical cytogenetic precedures. i find lots of good metaphases in the microscope and then i start the mISH experiment. But when i want to focus to the visible light in the microscope i can not find any of them. in witch step might i lose them. Generally i use my slides in different concentrations of SSC, In NAOH for denaturation, in ethanol series(70%,95%,100%) and in SSC with 0.05% Tween
Try to use formamide in 2x SSC for denaturation instead of NaOH. And dehydrate your slides in throughand ethanol series before you start with the FISH procedure.
you could try co-denaturing the dna and probe by using a thermobrite. if you don't have a thermobrite, you can use a hot plate. We co-denature probe and DNA at 72c for 3 minutes and hybridize over night or up to 48 hours. I can send a protocol if you'd like.
NaOH is probably too stringent to use it with FISH preps... switch to formamide in SSC as proposed by Sabine. Also, test your heating (hybridisation) plate for accuracy of set temperature to avoid overheating the slides (the same goes for temperature of washing buffers). Nevertheless, try age the slides at 60°C for short time (up to 30min) before proceeding to applying mFISH probes. And lastly, do not search for metaphases with visible light but counterstain them with DAPI and then search for them under appropriate fluorescent filter.
Hi
If you want to locate your mets in visible light initially, try 10X, with condenser diaphragm almost closed as these will be like unstained preparation. It is a good idea to locate them in DAPI staining using fluo. rather than visible. Also you can take stage vernier reading / England Finder reading to make sure you are observing spot where mets were there. You may observe a fluo. stained like DAPI stained metaphase preparation to make sure your focus, filter, etc. are OK, and then put your M-FISH slide. It is imp that your Hg bulb is 100Watts and not 50, and the objectives are 'neofluor' or Planapo or atleast Apochromat and not simple, including 10X or 20X for initial viewing or else visibility is easily compromised.
Regarding M-FISH hybridization process, my experience is, if you go by manufacturer's protocol, it is sure to work - no need for any modification. Vysis M-FISH kit comes with all the do's and don'ts regarding ageing, treatment etc.
I agree with Sabine. However, the handling of all washing especially with tween 20 and SSC can determine how well the spread cells can stay of peel off. The sample material may require very gentle washing. Sometimes, different materials respond to handling in different ways.
hello in my experience I have also used NaOH in preparations act after leaving the reagent must be thoroughly washed in distilled water, air dry to observe the material using a phase contrast microscope, or low full intensity of the light microscope until you can visualize your materials using the 40X objective and ensure at every step of your protocol to observe the material on your slide about taking the coordinates of your gear to return to locate when you find yourself over your protocol hope you serve the council greetings
thank you everyone Ι will try your suggestions. I hope tο have better resaults
have you tried using slides loaded or prepared silane is preventing any type of sample off the glass slide as you comment on every step of your protocol observes a metaphase previously the you located with your coordinates and you'll know you step're losing your stuff Cheers
try to put the slides in the oven at 60 for 1 hour without using ssc then add the probe
Poly L-lysine is good to retain the chromosomes in the slide if your FISH protocol has to be very stringent. You have to pre-treat the slides with this solution at the beginning, before you made the chromosome preparation.
May be is a good option. is very usefull to me.
In my experience, i've always obtained good results with RNAse and light Pepsin pretreatment. In the hottest days, only a light pepsin pretreatment, on slides prepared in the morning, and pepsin treatmen after 2-3 hours on a hot plate (45°).
After treatment, 70%-80%-90%-100% ethanol and vacuum dry.
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