Hello,

Thanks for the advice.  I am posting on behalf of someone in the lab  
and I don't have all of the details.  However, from what I  
understand, she is doing a methanol fixation.  I would prefer  
formalin, but she has been doing methanol for all her other staining  
procedures with these cells.

Why is a neuron specific tubulin necessary, and is it for a cell line  
or primary cells?


Caroline



On Aug 14, 2007, at 2:37 PM, Carl Hobbs wrote:

> Hi.
> you don't mention what fixing solution/ duration you use......
> I have not used the primary Ab you mention: however, as it is not a  
> neurone-specific Ab, you need to use another neurone-specific Ab to  
> confirm that what you are demonstrating are neurones ( sure,  
> morphology helps but is not neccessarily definitive) OR switch to a  
> neurone-specific beta tubulin ( beta III tubulin- neurone specific).
> Otherwise , it seems to me that your technique is about right,  
> provided that you are permeabilising after fixation but before  
> primary Ab incubation. You will need to permeabilise to optimise  
> staining, dependant on all the other variables in your technique,  
> that you haven't mentioned
> NB: Can't comment on appropriate dilution of your primary Ab but  
> your Alexafluor 488 should be fine......altho' I use it at 1/1000  
> dilution factor.
> Have a look at IF pics of neurone -specific beta tubulin here, for  
> example : http://www.immunoportal.com/
> Best of luck
> Carl
>
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