Immunohistochemistry Fixation - Follow-up...
khatipovNO at NOuchicago.edu
Wed Oct 17 12:27:32 EST 2001
I totally agree with you, D.K. I am always very skeptic to any data obtained
by immunostaining, and I believe that this method should be used to confirm
something that has been found before using more precise methods. If you know
what to look for using immunostaining, you can "adjust" your technique very
easily to fit the results you want.
"D.K." <dk at no.email.thankstospam.net> wrote in message
news:9qjsdi$ec6$1 at news.doit.wisc.edu...
> "Emir Khatipov" <khatipovNO at NOuchicago.edu> wrote:
> >By the way, could you share with us what is the protein you are staining
> >for? Is it a transcriptional regulator? In my experience,
> >gives conclusive results only if the protein of interest has specific
> >localization, like if it accumulates in intracellular compartments, or
> >complexes, etc., that can be visualized as foci.
> In my experience immunofluorescence is the least trustful method of
> all which can be used to "prove" absolutely anything. This thread is
> just a classic example of it:
> Melissa has some preconcieved idea and goes to a great lenght
> agaist obvious experimental result (non-nuclear localization) all the
> while getting excited "small proportion" showing "right" staining and
> not making proper controls (which would include doing _parallel_
> experiments with cell line for which nuclear staining is shown in
> the literature).
> >"Melissa Greeve" <gliftor at hotmail.com> wrote in message
> >news:350b1ac.0110152053.1a892b2c at posting.google.com...
> >> Hi all again,
> >> Just to let you know that I stained a couple of slides yesterday,
> >> trying two different fixation methods to show that my (thought to be)
> >> nuclear protein is indeed localised to the nucleus of cells grown on
> >> these chamber slides.
> >> As you may recall, I had been fixing the slides in 95% ethanol for 5
> >> minutes, but could only show the protein of interest to be
> >> cytoplasmic, suggesting leakage of the protein from the nucleus.
> >> Yesterday I tried fixing one slide in 95% ethanol (this time chilled
> >> to -20 degrees C) and the other in 4% formaldehyde in PBS (no
> >> sucrose), both at room temperature for 5 minutes. The ethanol-fixed
> >> slide again showed cytoplasmic and perinuclear localisation, with
> >> slight damage to the cells (blotchiness). However, the 4% formaldehyde
> >> fixation resulted in cytoplasmic localisation of the protein in most
> >> cells, but a small proportion showed distinctive nuclear staining
> >> (yay!), with no visible damage to cell structure. The staining was,
> >> however, more faint than I have previously observed (in one of those
> >> odd occasions where you get a fabulous but non-repeatable result), so
> >> I'll try a slightly less fixation time...
> >> Thanks all for your responses.
> >> Cheers,
> >> Melissa Greeve
> >> PhD Student
> >> University of Western Australia
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