Immunohistochemistry Fixation - Follow-up...

Emir Khatipov khatipovNO at NOuchicago.edu
Tue Oct 16 15:01:05 EST 2001


Melissa,
Did you permeabilize the cells after formaldehyde fixation? That is to my
knowledge what many people do. After washing off formaldehyde (or
paraformaldehyde) you can cover the cells with permeabilization buffer (see
below) for 5 min and wash the cells again to proceed with further
immunostaining. My understanding is that ethanol, in addition to fixing
cells, also permeabilizes them, whereas formalin does not, or does to a much
lesser extent than ethanol. Thus, if you had a weaker signal with
formaldehyde, you might have a better result if you permeabilize the cells
after fixation.

Permeabilization buffer (50 ml):
Triton X-100                            0.25 ml
1M Hepes, pH 7.4                    1 ml
5M NaCl                                    0.5 ml
1M MgCl                                    0.15 ml
2.5 M Sucrose (42.8 g/50ml)       6 ml
H2O                                            42 ml

By the way, could you share with us what is the protein you are staining
for? Is it a transcriptional regulator? In my experience, immunomicroscopy
gives conclusive results only if the protein of interest has specific
localization, like if it accumulates in intracellular compartments, or forms
complexes, etc., that can be visualized as foci.

Emir


"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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