Temperature for fixing cells in immunofluorescence microscopy - (May/24/2007 )

Hi everyone,
I am just wondering at what temperature you fix your cells for immunofluorescence microscopy. Some protocols recommend RT, some 37 and some 4. And for how long? I am confused
Thanks

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To fix cells, we do it at RT.

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For normal IF with PFA fixation, usually the process (including fixation) happens at RT; for methanol/acetone: usually on ice. However, depend on your experiments, sometimes it is necessary to fix at different temps. Also, the fixing solutions are always kept at low temp (PFA: 4oC, methanol/acetone: -20oC). Fixation periods are also depend on the fixing method: PFA: 10-15min while methanol is usually shorter: 5-10min. You can do longer fixation, but you always need to wash many times, as clean as possible so that you don't have high background later.

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it depends on the cell type. if your using suspension T or B cells gentle fixing at 4*C is recommended else RT is sufficient.

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we fix at RT for 10 min (various cell lines)

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Thanks eveyone for sharing your experience

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RT works for me, 10-20 mins... Interestingly room temperature varies from 15-40 degrees depending on where you are in the world and the state of your airconditioning...

D

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