Am 09.03.2010, 21:29 Uhr, schrieb Joel Schneider
<[email protected]>:
Is there a reliable way to perform IHC on cell monolayers without
fixation?
I am not experienced in IHC and but I've had success in using fixatives
such
as PAF for various staining protocols. Does anyone have a protocol
they'd be
willing to share that doesn't require fixation?
That depends on whether your antigenic site is extra- or intracellular.
For intracellular sites you need to permeabilise the plasma membrane with
acetone/methanol 1+2 at -20 °C for a few minutes.
In general, the fear of fixation in IHC is exagerated. With antiserum one
can ignore that issue completely, with monoclonals I would do some simple
testing to see if there is a problem. If the usual
formaldehyde/glutardialdehyde fixative does lead to problems, try an
aldehyde-free fixative, I had good success with Zenker's solution. This
has the interesting additional property of permeabilising the plasma, but
not the nuclear membrane. It can therefore be used to monitor cell
division. Note however, that it needs to be disposed of as toxic waste (Hg
and Cr).
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