Re: [Histonet] My Negative is now Positive ... IHC assistance needed?

From:"Andrea T. Hooper"

 From what I understand from reading and through my own personal 
experience, over time this Chrompure Rabbit IgG reagent from Jackson 
gives background.  I am not sure why. To avoid this I have heard 
people say they aliquot and freeze it and only keep a working aliquot 
at 4 deg C. I have this problem so much so with the Chrompure 
Reagents that I am considering looking for another source for my 
control IgGs.

>I recently worked up a new antibody for my lab.  It is a rabbit
>anti-mouse (antigen).  The antigen is found throughout the kidney and has been
>pinpointed by ISH to be located in the proximal tubules.  For the IHC,
>I have determined the best concentration for my antibody was 7 ug/ml. 
>I used Jackson's ChromPure Rabbit IgG, whole molecule as a negative
>control at the same concentration, diluted in the same buffer as my
>primary.  I am also using the Envision system by DAKO. 
>Lately I've noticed a lot of background coming up with my negative.  I
>re-ran a titre for my primary and had negatives at the corresponding
>concentrations.  To my surprise, I got a lot of background on the
>negative, and moreover, it appears to be decreasing as the dilutions get
>OK -- what's going on?  Have I contaminated my negative somehow??  I
>keep the glass jar it originally came in at 4 degree C.  When needed, I
>aliquot what I need under the Biosafety Cabinet (sterile conditions). 
>If it has been contaminated, what has it been contaminated with and why
>is it giving me background?  How can I avoid this in the future (can it
>be aliquoted and frozen)?  Other suggestions will be very helpful.
>My positive looks good.  Very clear staining and background only seen
>when Ab concentration is very high.  I need a clear negative though to
>support my primary Ab results.
>Thank you all again for your help.


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