I am planning to do IFA to see the intracellular localization of a protein in Leishmania parasite. Is it necessary to do IFA just after fixation of cells on slides or I can store these slides for future use. Does IFA result varies in stored cells from freshly coated cells?
I usually fix the cells and do the IF after. i keep them at +4C in PBS for 1 week maximum. You could add sodium azide to PBS.
Hi, Kumar, after proper fixation you can keep cells at 4°C for soem time, at least 1-2 weeks is OK. You do need to use immediately.
I usually fix the cells and do the IF after. i keep them at +4C in PBS for 1 week maximum. You could add sodium azide to PBS.
Hi Kumar,
It is advisable to use fixed cells for IFA as soon as possible, becuase the fixation apparently modifies cellular morphology to some extent. Also it depends upon the fixative you use. i usually use PFA in PBS,it last quite longer than other fixatives.
Obviously it depends on the purpose and always is advisable to use the cells as soon as possible. However, properly fixed Leishmania promastigotes can be frozen (-20ºC) for months and then used for diagnosis (e.g. in dogs).
Hi, I've never used Leishmania, but I have quite a lot of experience in IF in mammalian cells. You can keep the sample at 4 degrees after fixation, but the sooner you do the IF the better results you get (in term of quality of the staining and backgroud). I'd suggest you to plan the experiment in a way that allows you to do the IF straight after the fixation (or a couple of days afterwards).
Best regards,
Elena
Just remember not to Permeabilize! Fixed cells will tolerate storage in PBS for most antibodies. Unfortunately- fresh is best... But if you have a good antibody, it should be ok. In my experience- it is not optimal to delay IF for cytoskeleton associated proteins.
I have no experience for Leishmania, but tissues tend to loose reactivity during wet-dry cycles. "Wet" also means precipitation of humidity when a cold slide is quickly transfered to a warmer place!
Parasites fixed with acetone and store at 4oC , dried can be used until 30 days.
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