Hi,
I am going to do an immunofluorescence experiment. I went over my protocol and it says that I have to do methanol fixation. I would like to know why I have to do serial ethanol rehydration steps (100 -> 95 -> 75 -> 50) after fixing cells with methanol? is there any other way to rehydrate cells? I will appreciate help.
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#2
bob1
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Thelymitra pulchella
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Posted 25 August 2009 - 04:53 PM
You need to rehydrate as the methanol shrink the cells through dehydration. If you want to avoid this use s different fixative such as paraformaldehyde.
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minylim
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Posted 14 September 2009 - 03:53 PM
I agree... and I heard paraformaldehyde or formaldehyde works much better than methanol...
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fluffybunny
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Posted 08 October 2009 - 03:24 AM
Hi bompu,
rehydration is for reintroducing liquid back into the cells (viable) so that ur epitopes of whatever protein you're detecting can be bound by antibodies. During fixation, all water are taken out completely from your cells. Serial rehydration is the only way i suppose. I do ethanol rehydration (100 -> 90 -> 85 -> 80) then wash my cells with PBS.
why do u fix cells in methanol? is it the most suitable fixing agent for immunofluorescense?
rehydration is for reintroducing liquid back into the cells (viable) so that ur epitopes of whatever protein you're detecting can be bound by antibodies. During fixation, all water are taken out completely from your cells. Serial rehydration is the only way i suppose. I do ethanol rehydration (100 -> 90 -> 85 -> 80) then wash my cells with PBS.
why do u fix cells in methanol? is it the most suitable fixing agent for immunofluorescense?
