Hi, I want to karyotype my mESC lines for characterization. I used Giemsa staining earlier, without arresting my cells in metaphase, therefore now finding it quite hard to find countable chromosomes within the fixed-stained slides. I was hoping if I could also try C-band staining for my cell lines. Now that I have many samples already fixed in KCl- Carnoy's fixation sol., can anyone please suggest any other stain that I could use instead of Giemsa, which could make the counting easier and more accurate- as sometimes its difficult to distinguish between chromosomes that are close to each other.. Thanks a lot!
Hi Pooja, I would recommend G-banding because it is an easier, faster, simpler technique than C-banding. Plus, it gives you the option to identify chromosomes if needed. I don't do a lot of mouse chromosomes but in the ones I have done the centromeres are very prominent with G-banding. I prefer Leishman's stain over Giemsa but I don't think that matters too much...
Hi David,
Thank you for your suggestion.
Could you perhaps share the protocol for the same? Would the fixation protocol also vary accordingly? Thanks again!
Hi Pooja, I just sent you a G-banding protocol. If you are using a KCL hypotonic - Carnoy's fixation method that is the same that I use...
Hello, Pooja. If you have fluorescent filter on your microscope you may try DAPI staining. On mouse chromosomes it will have brighter centromeric regions that may help with counting.
Dear Polina, Thanks for your tip. Shall definitely try it out! thank you. :) X
Hi all. I am attempting to establish a protocol for quick analysis of mESC karyotype and am interested in the DAPI staining technique. What is the timeframe from cell prep to karyotype analysis? Would you be willing to share protocol? @Pooja Khurana @David Mcdonald @Polina Perelman Thank you, Mike
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