Hello Everyone,
For a time-course experiment, in which I need to collect samples everyday for bio imaging using DAPI staining, I'd like to know if there is a way in which I can fix my cells in the slide with the DAPI for later analysis after I collect all my samples.
My experiment lasts 7 days.
Thanks for you help in advance :)
Hi Tatiana,
For your purpose, the simpler the better : fix your sample in 4% formaldehyde in potassium phosphate buffer 0.1M pH 7.2, Triton x-100 0.1%
As your specimen is aqueous I suggest you to put 1mL 2X fixative solution (8% formaldehyde in 0.2M KPi) in 2 mL tubes, then add 1 ml of your cell suspension. Let the fixation occur on the bench.
You can add DAPI at the step you want, but I suggest you to wash your cells before mounting them in glycerol mounting medium.
Cheers
Oliv
Hi Oliv,,
Thank you so much for your suggestion. Does this prevent any metabolic changes in my cell suspension? Also do you have the link to any publication that applied this method?
I'm definitely going to try it! Thank you so much.
Cheers,
Tatiana
It is a standard method for fixing biological sample.
I found this article on Chlamydomonas :
doi: 10.1105/tpc.114.129270
Hello Tatiana,
I had experience with time course experiments for DAPI staining. In my case, I worked with mosquito follicles, and after dissection, I stored tissues in10% Neutral buffered formalin (NBF) for 24h at RT, then for long term storage, I changed the solution with 70% ethanol and stored at 4 degree. With that preservation, I never had any issues in the integrity of the images while I captured all my images at the same time.
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