What is the problem, could you please be more specific?

Thanks for the attention

I have two question:

1) I must analyze metaphase cells (to see the chromosomes) or interphases cells

2)How to do the fixation of the cells and how to drop the cells on the slides before the denaturation?

Thank you once again

Tamar Tsertsvadze

1) Usually you get both on your slide, adherent cells without synchronisation gives around 2-3% of metaphase spreads, rest is interphase nuclei, so for a good statistics use as much cells as possible. Your protocol suggest, that for your purpose [detection of deletion] interphase nucleus is enough.

2) You need to detach cells, fix them in suspension [we use MtOH: Acetic acid 3:1 after hypotonic treatement- just spin the cells and change solution for fixative] and than put a drop [30ul?] of cells in fixative on clean slide from a few cm distance [10?:)]. Let it dry. Its adviced to make slides at least a day before fish procedure. You can always check how dense they are [regular phase contrast should do]. I havent found any good protocol for keeping cells attached during protocol, however i was aiming for metaphase spreads, so maybe in your case you could leave them attached?

3) Your protocol is somehow a bit simple, if it doesnt work I can provide with our protocol [but for suspension cells].

W.

Thank you very much for this exhaustive answer, You help me very much and answer all my questions.

I'm still trying to do acourding this protocol, if it doesn't work Then I will need you help

Tamar

If you send me your protocol, I'll do it together in parallel

Thank you once again

Dear Wojciech Witarski

I done Fish according my protocol ( metasystem)

but at last when i look slide under the microscope have not get result, I think Dapi didn't paint cells

can you help me?

What can be reason?

What could be the reason that the dipi can not contain cells?