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Negative control is positive for IF staining


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#1 Koi

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Posted 05 December 2010 - 12:57 PM

Hi,

I have been getting positive staining for my negative control (by omitting primary antibody or replacing primary antibody with normal serum).
I am using indirect immunofluorescence technique trying to stain tissue sections of turkey brain. The tissues was fixed with 4% paraformaldehyde.
For negative control sections, I omitted primary antibody but still get the staining from the secondary antibodies. I used Donkey anti-rabbit FITC, Donkey anti-goat FITC from JacksonImmunoResearch and Goat anti-rabbit FITC, Donkey anti-goat FITC from Santacruz. I blocked the sections with normal donkey serum in the first step (tried both 5% and 10% for 30 min with and without BSA). I incubated secondary antibody for 1 hr in dark. Does anyone know what can possibly cause positive staining when there is no primary antibody? I called it positive staining because it is not the background staining and it stains only on the specific area that I am interested in. I know there are many types of cells surrounded in the tissue section since I use the whole brain sections. But, all those cells in different areas did not show any staining.
There is no autofluorescence in that area.

Any ideas would be appreciated.

Thanks,
Koi

#2 sgt4boston

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Posted 08 December 2010 - 05:32 AM

Is your conjugate diluted sufficiently...lower concentration?
Can you block with non-specific IgG of the same species as the secondary antibody?
Are the wash steps sufficient or stringent enough?

#3 tadala

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Posted 31 January 2011 - 01:34 AM

I can't help, but I have the same problem (which I have to solve), but don't know how, at least yet.
I am going to use differnet methods of cell fixation for the good begining.

#4 Koi

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Posted 31 January 2011 - 06:41 AM

It was autofluorescence in my case. I did not think of it before since we have been working with this area for years with no problems. You can try checking your sections before staining. If that is the case there are many ways to fix it. Good luck!




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