NFkB nuclear staining problems - (Apr/02/2007 )
Hi everybody,
I'm doing some experiment un nuclear translocation of NFkB in rodent
mesangial cells. First I was using a 4% parafomaldeide + 0,1% 15'
Triton fixation method and I observed a nuclear staining also in basal
conditions (as a stimulation I use high levels of glucose), then I used
a 100%-10' -20° methanol method (without triton) and I observed no
nuclear staining but also in the stimulated condition. Using both
fixation methods I made also an LPS stimulation and observed a
translocation with each one. Can someone explain me why there is such a
difference in staining with different fixation methods and why with PFA
I see a nuclear staining in stimulated condition and with methanol no
(but yes with LPS stimulation)? Thanks
Fox71
Additional information: I tried also with less time of triton (2'
instead of 15') and saw less nuclear staining in basal (and less in
stimulation) but always less than with methanol.
The difference isnt between your fixatives exactly but more your epitope retrieval/permeableisation in relation to your fixatives. the reason for using the triton x on PFA is to break the cross linking which would reduce the staining - its also used to permeableise the nucleus to better your nuclear staining (in other techniques) - methanol fixation requires less retrival but i'd still use a low level one (ie the triton x - the alternatives involve pressure cookers, microwaves and digestion).
a good fixative is ten minutes in acetone:methanol (50:50) then 15 min 0.2% triton x - i use this on frozen sections and it comes up a treat - cells shouldn't be much different (keep the fix in a -20 freezer)
a good fixative is ten minutes in acetone:methanol (50:50) then 15 min 0.2% triton x - i use this on frozen sections and it comes up a treat - cells shouldn't be much different (keep the fix in a -20 freezer)
Thank you Dominic for the explanation, in these days I'll try your fixation method.
Just another question (sorry but I'm not an expert on these kind of techniques): is it possible that an excessive permeabilization of the nucleus can give an excessive apparent staining , in other terms if it can change the results?Thanks again
good question
epitope retrieval in wax (fixed in PFA) sections is a case of doing as much damage as is possible without removing the tissue from the slide ie there is a fixed maximum you cant go beyond. this is because you are retrieving epitopes not creating them. its the same with antibodies you cant lable more than there are. so, no you cant over-permeablise.