Hi Everyone,
I could not agree more with Glen's comments. The more I see different immunofluorescent staining protocols (and the questions they raise), the more I realise that most researchers do not even question why they do a particular treatment. Fixation and permeabilisation techniques are good examples of "chinese whispers" where the technique gets handed from one person to the next without question.
The choice of fixative (and the time and temperature of fixation) can be critical in determining if a particular antibody works at all. Formaldehyde (depolymerised paraformaldehyde) and methanol are commonly used - depending upon the location and shape of the epitope to be examined, they can give vastly different apparent affinities of the antibody.
I think an important question that is almost never asked is "How long should I fix for?". I'm sure most people try a time suggested by someone else and, if they get some staining, never change or test their protocol further. Similarly, "what concentration should I use?" is frequently asked.
I would highly recommend that everyone read the following article - I'm sure it will provide "food for thought".
"Formaldehyde, Formalin, Paraformaldehyde and Glutaraldehyde: What They Are and What They Do." by John Kiernan, Microscopy Today, January, 2000, pages 8 - 12.
...rant finished :-)
Cheers to all
Paul
Dr Paul Rigby
Associate Professor
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley WA 6009
Phone (61 8) 9346 2819
________________________________
From: Confocal Microscopy List on behalf of Glen MacDonald
Sent: Thu 20/05/2010 12:57 PM
To: [log in to unmask]
Subject: Re: Please help
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
>>
> **********************************************************
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