To continue, whenever I tried a new antibody I would always do a
fixation and titration analysis ([fresh]PFA, GA, methanol, ethanol,
nothing). Yes, it takes time and costs money in reagents, but at least
you don't have to second guess that part of your assay from there
forward. Sometimes you are surprised at the results. Certain antibodies
work poorly with certain fixatives. 

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872
http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
ing/Pages/default.aspx

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]]
On Behalf Of Paul Rigby
Sent: Thursday, May 20, 2010 10:45 AM
To: [log in to unmask]
Subject: Re: Please help

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
>>
> **********************************************************
> 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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