IHC protocol

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Wed Mar 10 09:07:54 EST 2010


Am 09.03.2010, 21:29 Uhr, schrieb Joel Schneider  
<joelschneider914 from gmail.com>:

> 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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