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immunofluorescence - antibody choice for double staining


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#1 vetticus3

vetticus3

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Posted 28 November 2013 - 04:57 AM

Hi,

 

I need some advice - I'm going to be doing a double IF staining on PFA fixed cells.  One antibody I have - anti rabbit... the other antibody I'm going to buy.

The problem is the best antibody is also anti rabbit. 

There is an alternative that is anti mouse, but it has only been tested (and guaranteed) in IHC.

So... should I buy the anti mouse that may or may not give a result?

or

should i buy the anti rabbit and do a full stain with 1 ab, and then repeat with the second (which i haven't done and i'm not confident that it would work)?

 

Opinions?

 

V



#2 bob1

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Posted 28 November 2013 - 12:25 PM

I would go with the mouse.  If you do the double rabbit, unless you can guarantee that all the first antibody is bound by secondary, then you will get some false binding.  The other way around this is to buy pre-conjugated primaries or to do it yourself (expensive).



#3 vetticus3

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Posted 29 November 2013 - 02:01 AM

That was my initial line of thinking...  thanks for the advice.

Best,

V






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