Immunohistochemistry with C2C12 cells

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Mon May 31 12:33:08 EST 2004


Don Incognito wrote:

> Greetings,
> 
> Has anyone experience with immunohistochemistry in the C2C12 skeletal
> muscle myoblast cell line? I've had no luck. I've tried fixing with 4%
> PFA for 5 - 10 minutes, pre-blocking with 2% BSA or normal goat serum
> (5% and 10%), permeabilization with acetone or PBS-T for 10 minutes,
> incubation with primary antibody at 37 degrees for an hour or room
> temperature overnight (haven't tried a 4 degree incubation, but does
> that make a difference?), and various secondaries, but haven't been
> able to raise a reliable non-background signal. 

I would use the blocking step after permeabilisation.

The main difference between incubation at 37 degrees, 1 hour and 4
degrees over night is that with the latter it is possible to recycle the
antibody solution. With the small volumes used in histochemistry, that
is not important, but on blots that can be important.

Apart from those general remarks we would need to know a couple of thing
to give you suggestions:

Are you using monoclonal antibodies or an antiserum as primary?
Monoclonals bind to one specific epitope of the antigen, if that is
blocked no binding is possible. Polyclonals are less sensitive.
Formaldehyde is a cross-linking fixative, which can nail an epitope if
you are unlucky. You could try different fixatives, I had good results
with Zenker's solution in a similar situation. Chromate or osmium
tetroxide based fixatives could also work.

Is the antibody raised against native or denatured antigen? Often
antigens are purified by SDS-PAGE befor immunisation. The resulting
antibodies (again especially monoclonals) may not bind to the native
protein.

What exactly do you mean by "permeabilization with acetone or PBS-T"?
PBS, even with a small amount of Tween will not permeabilise the cell
membrane to make cytosolic antigens accessible. The best results are
obtained with aceton/methanol mixtures at -20 degrees celsius (mixture
needs to be adjusted to the problem, 3+2 is a good starting point).

Do you have positive and negative controls (cells with and without the
antigen in question, treated exactly as the experimentals)? Without the
former a negative, without the latter a positive result is meaningles.




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