Dear Olga,
the article to which I referred:
Melan and Sluder (1992) J. Cell Science.

The question of rearrangement will depend upon how well  the antigens are bound to other proteins and resistant to solubilizing.  Triton can be very helpful in maintaining proteins in solution to prevent precipitation and non-specific binding.  Some antibodies are more susceptible than others.  In a polyclonal antisera if some antibodies are sticky its probably not so big a problem as with a monoclonal where such problems affect the entire population of molecules. 

How are you are labeling the proteins and observing them?  If using fluorescence, I'd avoid glutaraldehyde due to its high fluorescence. But Mario is correct that is may help if you are using brightfield labels.   

> I am working with tissue culture cells.
> FLAG tagged ectopic protein and endogenous one are colocalized very well.
How was this determined?  
> I will definitely try to do only PFA, without Triton.
Yes, and maybe also try 1% or 2% PFA, and try cold methanol as suggested.
> However, everybody who responded, told we that the old protocol (4% PFA, 40min, then Triton 0,025% in every following blocking and washing solution) did not reveal nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton.
That is very possible, but without testing it is only an opinion consistent with "what everyone else does".
> My new protocols: PFA 4% (5-20min) followed by Triton 0.1 % (5-20min) - all combination shows identical results - cytoplasmic and nuclear staining both for the Protein ABs and FLAG
> Cel Signalling general protocol also calls even for more Triton - like - 0.5%. They do not use ABs which do not work  well in that general protocol.
> I was very happy about public consensus until your comments about Triton.
Most protocols from vendors are just as untested as any other published protocol.  Mostly taken from the literature, sometimes developed in-house with tissues known to produce a robust signal.  It worked - sweet, get some pictures!.  And often biased towards robust signals with short incubation times using high concentrations of primary and secondary antibodies to make the product convenient to use.

By the way, have you titered your antibody concentrations?  What do you see with substituting blocking solution for the primary antibody at the current dilution of secondary?

It is also possible that something else is affecting distribution of protein, such as culture conditions.  Could the perinuclear distribution have been associated with organelles?  Also, are you using the exact same culture conditions and antibodies as in your prior lab?   

Regards,
Glen 
> Now  a new question has come up: Could my protein redistributed to the nuclei because of Triton in my new protocol???

> Which of these is TRUE??
> Thank you, 
> Olga
> 
> Or, vice versa. My protocol does something strange that relocates my protein to  nucleus.
> 
> Olga Makarova, PhD
> Research Specialist
> 5323 MSRB III
> 
> 
>>>> Glen MacDonald <[log in to unmask]> 5/20/2010 12:57 PM >>>
> Generally, PFA sufficiently permeabilizes for most immunolabeling.  PFA crosslinking may distort the antigen rather than block access.  A lot of protocols include Triton because they were inherited from someone else or that is just how someone learned to do it.  Many researchers never take the time to see if it can be eliminated or reduced to achieve results that are the same or better.  I don't have the reference at hand, but Triton has been shown to rearrange antigen distribution.  Other approaches with PFA tissue is to pass vibratome sections through graded sucrose into 30% sucrose then freeze at -80C.  thaw slowly in the refrigerator and return to PBS.  the ice crystals rip big holes in the membranes but the histology is much better than a frozen section.  This might work with cultured cells.  You could try shorter fixation, say 3 min. or 1% PFA.  a 3 min treatment (10 min for 40 um vibratome sections) with 1% SDS (an optimal concentration may be less) before the blocking step may also improve the labeling.
> 
> coagulating fixatives tend to preserve antigenicity better, such as cold methanol or acetone.  Carnoy's solution and fixes with picric acid are also options.  
> 
> Good luck
> Glen
> 
> Glen MacDonald
> Core for Communication Research
> Virginia Merrill Bloedel Hearing Research Center
> Box 357923
> University of Washington
> Seattle, WA 98195-7923  USA
> (206) 616-4156
> [log in to unmask] 
> 
> 
> 
> 
> 
> 
> 
> 
> On May 20, 2010, at 9:31 AM, Olga Makarova wrote:
> 
>> Thank you very much, I will you approach.
>> Olga
>> 
>> Olga Makarova, PhD
>> Research Specialist
>> 5323 MSRB III
>> 
>> 
>>>>> Artem Pliss <[log in to unmask]> 5/20/2010 12:16 PM >>>
>>> I guess , one of my questions is whether  with the old protocol they  did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton.
>> 
>> Answer is yes. It is difficult or even impossible to detect nuclear
>> antigens using such an intense fixation with PFA (4%, 40 min) followed
>> by permeabilization with 0.025% triton.
>> As a control you may also try 20 min fixation in methanol on ice (no
>> triton is needed). Methanol fixation optionally may be followed by the
>> immersion of cells in  acetone for 10 sec and air dry. Methanol does
>> not preserve the structure as good as PFA does, but it will reveal the
>> distribution of your protein more efficiently.
>> Art
>> 
>> On Thu, May 20, 2010 at 11:42 AM, Olga Makarova <[log in to unmask]> wrote:
>>> Mario, thank you very much for your response.
>>> 
>>> I am really confused about many things. I 've found many protocols for IF and all of them ask for both the PFA and Triton.
>>> My protein is a SEPT9 which is implicated in many cancerous transformations.
>>> In both protocols FLAG and protein staining colocolize very well..
>>> Sorry for the "opposite" I meant that instead of familiar perinuclear staining which was observed by people before, I see nuclear and cytoplasmic staining.
>>> I guess , one of my questions is whether  with the old protocol they  did not see the nuclear staining because of too much crosslinking by PFA and too little permeabilization with Triton.
>>> Or, vice versa. My protocol does something strange that relocates my protein to  nucleus.
>>> 
>>> Thank you,
>>> 
>>> Olga
>>> 
>>> Olga Makarova, PhD
>>> Research Specialist
>>> 5323 MSRB III
>>> 
>>> 
>>>>>> Mario <[log in to unmask]> 5/19/2010 3:01 PM >>>
>>> Olga,
>>> 
>>> In and of itself, paraformaldehyde does not typically require Triton
>>> as it is quite membrane permeable unlike glutaraldehyde. Adding 0.1 %
>>> Triton is still a fairly low concentration but maybe enough to punch
>>> holes in your cell membranes causing a complete redistribution of
>>> intracellular electrolytes (K+, Na+, Mg-, Ca-, etc.). Not knowing
>>> which protein you are tagging with FLAG, I can only speculate what
>>> the consequences might be.
>>> 
>>> It is a bit unclear what you mean by "opposite results." I am
>>> assuming that in the first protocol (low Triton) you get what appears
>>> to be colocalization of your protein and the FLAG tag, which is what
>>> you logically would hope for. In the high Triton protocol, you lose
>>> the peri-nuclear localization, but is this true also for the
>>> protein-FLAG version, i.e., do the distributions remain coincident or
>>> is that lost? If colocalization remains then one would be tempted to
>>> blame the high Triton for possibly mobilizing the target proteins
>>> away from the nucleus and into the cytoplasm.
>>> 
>>> If target protein and the FLAG version no longer coincide then things
>>> are more complicated.
>>> 
>>> Anyway, using PF without detergent should work fine for fixation and
>>> I would tend to believe it before accepting simultaneous PF and
>>> Triton. Did you try the PF for 40 min. then the higher 0.1 % Triton?
>>> 
>>> For sturdier fixation you might also try 3-4% PF plus 0.2%
>>> glutaraldehyde in the initial fix. The PF goes in first and does the
>>> initial fixation and allows glut. to follow and reinforce your target
>>> crosslinking.
>>> 
>>> With regards to using milk and BSA for blocking, I would seriously
>>> consider changing over to an appropriate animal serum appropriate to
>>> you antibodies, or better yet a product like Pierce's SuperBlock.
>>> 
>>> Let us know how things go,
>>> Mario
>>> 
>>>> Hello everybody,
>>>> 
>>>> This is my first message to confocal community. I am also molecular
>>>> biologist:-(( and do not know all tricks in IF.
>>>> I am trying to understand where is my FLAG tagged protein localized
>>>> in breast epithelial cells which also has endogenous one.
>>>> I grow cells in chamber slides.
>>>> Old lab protocol show strong perinuclear  staining for both ABs to
>>>> our protein and FLAG. Protocol include:
>>>> 40 min of 4% paraformaldehyde treatment (previously frozen!!) in PBS at RT.
>>>> Permeabilization and blocking solution: 5%milk, 1% BSA, 0.025%Triton
>>>> for 1 hour.
>>>> 
>>>> I was suspicious of too long paraformaldehyde treatment and too low
>>>> Triton percentage.
>>>> 
>>>> I tried fresh PF  5 and 20 min with Triton 0.1% for 5 and 20min and
>>>> get opposite result.
>>>> 
>>>> In my variation  our protein mostly cytoplasmic and nuclear.
>>>> 
>>>> We also know that under some condition this protein create
>>>> filamentous structure.
>>>> 
>>>> If somebody could help me to understand which protocol is right,
>>>> what kind of variation of protocol I could use to solve the problem,
>>>> I would very much appreciate.
>>>> 
>>>> Thank you,
>>>> 
>>>> Olga
>>>> 
>>>> Olga Makarova, PhD
>>>> Research Specialist
>>>> 5323 MSRB III
>>>> 
>>>> 
>>>> **********************************************************
>>>> Electronic Mail is not secure, may not be read every day, and should
>>>> not be used for urgent or sensitive issues
>>> 
>>> 
>>> --
>>> ________________________________________________________________________________
>>> Mario M. Moronne, Ph.D.
>>> 
>>> [log in to unmask] 
>>> [log in to unmask] 
>>> **********************************************************
>>> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues
>>> 
>> **********************************************************
>> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues 
> **********************************************************
> Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues