Hi everyone,
There are so any protocols for immunocytochemistry (ICC).
I usually use 3.7% paraformaldyde (PFA) for 15 min at Room temperature (RT) for fixation step.
But I recently saw some protocol in which they use only 2% PFA for 30min at RT.
What do you prefer?
Can you tell which one miht be better 3.7% or 2% PFA?
Thanks

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Hi,

I usually use 4% PFA for 15min at RT. Works absolutely fine. I have some antibodies which don't work if the tissue is "overfixed". So for staining with them, I use 2% PFA, but still only 15min...I don't really see a reason to use low concentrations of PFA and leave the cells on room temperature for that long.
Comments, anyone?

Thanks!

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Yes that is what I was wondering.
Maybe is to get a slower fixation process.
Is it better to fix quicker?

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Some fluorophores are more sensitive to fixation. I don't know which one you use (and I only use fluorescent proteins myself, so I'm not sure if this is true for non-protein-fluorophores), but for instance DsRED is more sensitive to fixation than for instance EGFP, meaning that upon fixation, DsRED loses quite a lot of fluorescence intensity whereas for EGFP the loss is less pronounced. Using lower concentrations of PFA might overcome this problem a bit, without loosing efficiency in fixation itself.

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I also use 4% for 10 min at room temprature

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